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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:10.)
© 2000 American Heart Association, Inc.

Vascular Biology

Oligoclonal T Cell Expansions in Atherosclerotic Lesions of Apolipoprotein E–Deficient Mice

Gabrielle Paulsson; Xinghua Zhou; Elisabeth Törnquist; Göran K. Hansson

From the Center for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden.

Correspondence to Göran K. Hansson, Center for Molecular Medicine L8:03, Karolinska Hospital, S-17176 Stockholm, Sweden. E-mail Goran.Hansson{at}cmm.ki.se

	Abstract

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Abstract—T cells are present in atherosclerotic lesions at all stages of development. They exhibit activation markers and are particularly prominent at sites of plaque rupture. This suggests that T-cell–mediated immune responses are involved in the pathogenesis of atherosclerosis. Antigen-specific T cells reactive with oxidized lipoproteins and heat shock proteins have been isolated from plaques, indicating that local activation and clonal expansion might occur. To analyze different stages of atherosclerosis, we have used a murine model. Targeted deletion of the apolipoprotein E gene results in severe hypercholesterolemia and spontaneous atherosclerosis, with lesions containing large numbers of T cells and macrophages. We have analyzed mRNA for T-cell antigen receptors (TCRs) from aortic fatty streaks, early fibrofatty plaques, and advanced fibrofatty plaques of such mice. Polymerase chain reaction amplification of complementarity-determining region 3 (CDR3 region) of TCRs was followed by spectratyping of fragment lengths. This analysis detected all types of variable (V) segments with a gaussian distribution of CDR3 in lymph nodes. In contrast, a restricted heterogeneity was found in atherosclerotic lesions, with expansion of a limited set of Vß and V segments and a monotypic or oligotypic CDR3 spectrum in each lesion. Vß6 was expressed in all lesions; Vß5.2, Vß16, V34s, and V9, in the majority of lesions; and Vß6, Vß5.2, and V34S, in lesions at all 3 stages of development. The strongly skewed pattern of the CDR3 region in the TCR is indicative of oligoclonal expansions of T cells and suggests the occurrence of antigen-driven T-cell proliferation in atherosclerosis.

Key Words: atherosclerosis • antigen receptors • rearrangement • T-cell antigen receptors • hypercholesterolemia

	Introduction

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Immune mechanisms play an important role in atherosclerosis.^{1 2 3 4} Monocytes and T cells enter lesions at a very early stage; this process is governed by adhesion molecules and chemokines induced in hypercholesterolemia. The first stage of atherosclerosis, the fatty streak lesion, is largely composed of fat-filled macrophages together with some T cells. Transformation of this lesion into a true fibrofatty atherosclerotic plaque is associated with smooth muscle immigration and the formation of a fibrous cap around the macrophage core. In advanced lesions, activated T cells and macrophages are also important components that secrete cytokines, such as interferon-, interleukin-2, tumor necrosis factor-, and interleukin-1, all of which have been shown to modulate gene expression in vascular endothelial and smooth muscle cells.^{2 4 5} Finally, inflammatory activation in the advanced plaque leads to metalloproteinase secretion, collagenolytic activity, plaque rupture, thrombosis, arterial occlusion, and infarction.⁶ This clinical event is associated with the release of inflammatory acute-phase reactants such as interleukin-6 and C-reactive protein (CRP) into the circulation and also with increased levels of circulating activated T cells^{7 8}

The presence of activated T cells suggests that specific immune responses may occur in the plaque. Several candidate antigens have been detected in human and experimental plaques, including oxidized LDL, heat shock proteins, and microbial antigens, such as Chlamydia pneumoniae proteins.^{9 10 11} In fact, a substantial proportion of CD4⁺ T cells isolated from human plaques recognize oxidized LDL as an HLA-DR restricted antigen.¹² This leads to cell division as well as cytokine secretion, and one would therefore expect to find clonal expansions of antigen-specific CD4⁺ T cells in plaques. However, this was not confirmed in previous studies of advanced human plaques with the techniques of limited resolving capacity that were available at that time.^{13 14}

An inflammatory process will recruit T cells irrespective of their immunologic specificity; therefore, a chronic inflammatory lesion usually contains a heterogeneous T-cell population. This does not rule out the possibility that chronic lesions contain detectable clonal expansions of T cells at different stages of development. To address this issue, we have analyzed T-cell antigen receptor (TCR) mRNA in atherosclerotic lesions at different stages of disease development. Our data, which used high-resolution analysis of the antigen-binding regions, show the TCR usage to be highly restricted and strongly skewed in both fatty streaks and fibrofatty plaques of apoE-knockout (apoE-KO) mice. This is indicative of oligoclonal expansions of T cells and suggests the occurrence of antigen-driven T-cell proliferation in atherosclerosis.

	Methods

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Animals
The mice used in the present study were the progeny of apoE-KO animals that were initially created by homologous recombination in embryonic stem cells¹⁵ and backcrossed to the C57BL/6 strain for 6 generations. Male mice were weaned at 4 weeks and put on cholesterol-containing diets at 8 weeks of age. One group of mice were fed a "western" diet containing 0.15% cholesterol, and the other mice received a high-cholesterol diet with 1.25% cholesterol and 0.5% cholic acid. Diets are described in detail in Reference 3³ . These 2 types of dietary treatment result in serum cholesterol levels of 15 to 20 mmol/L (western diet) and 40 to 50 mmol/L (high-cholesterol diet), respectively.¹⁶ Diet and water were provided ad libitum. All experiments were approved by the regional ethical committee for animal welfare.

Time Schedule and Sample Collection
Groups of 3 or 4 mice were killed after 10 or 18 weeks of diet treatment by exsanguination under carbon dioxide anesthesia. After PBS perfusion, both lymph nodes and atherosclerotic lesions from the proximal aorta were carefully dissected out and snap-frozen in liquid nitrogen. Segments of aorta were snap-frozen in OCT compound for cryosectioning.

Immunohistochemistry
Cryosections were processed for immunohistochemistry as previously described^{3 16} with minor modifications. Briefly, rat anti-mouse CD4 and CD8 (PharMingen) were applied to acetone-fixed cryosections. After a washing step, biotinylated goat anti-rat IgG(H+L) (Dakopatts) was applied, followed by avidin DH/biotinylated peroxidase complex (Vector Laboratory). Controls were stained with irrelevant monoclonal antibodies or without primary antibodies. Sections were counterstained with hematoxylin.

mRNA and cDNA Preparation
After a brief perfusion with PBS, the heart and proximal aorta were dissected out from apoE-KO mice (for treatment, see above) and placed in ice-cold PBS. The atherosclerotic lesions were isolated within 1 hour in under a microscope and frozen immediately into liquid nitrogen. Samples from each mouse (m2 to m7 and m11 to m13) were kept individually (high-cholesterol diet for 10 and 18 weeks, western diet for 18 weeks) except samples 8 to 10 (western diet for 10 weeks), to which lesions from 3 mice were pooled because of the small size of the lesions (Table 1). Aortic lesions and lymph nodes were homogenized individually in a Dismembrator (B. Braun Melsungen AG) while they were still frozen. Lysis buffer (Dynal) was added to the homogenate, mRNA was isolated on oligo-dT–conjugated magnetic beads (Dynabeads, Dynal), and single-stranded cDNA synthesis was performed by using Superscript II (Life Technologies) and random pdN6 primers (Pharmacia) in the presence of Rnasin (Promega).

View this table: [in this window] [in a new window]   Table 1. Plaque Samples From Aortic Root of apoE-KO Mice Used in Spectratyping RT-PCR Analysis

PCR Reactions
cDNA, which was analyzed from each sample individually (Table 1), was amplified by polymerase chain reaction (PCR) in a master mix containing 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 1 mmol/L dNTP, and 0.2 U/mL Taq polymerase (Life Technologies).¹⁷ For amplification of 19 TCR chain cDNA, a set of 19 V primers was used together with 1 C primer that had been tagged with FAM fluorochrome (Genset). Similarly, 24 Vß primers were used with 1 fluorescent Cß primer to amplify the TCR Vß chain cDNA. Final concentration of primers was 0.2 µmol/L each. All primers are listed in Table 2. Primer sequences for V, Vß, C, and Cß were used in the reverse transcriptase (RT)-PCR reaction. All sequences were taken from References 17¹⁷ to 32. Each reaction was performed in a separate tube; ie, each PCR tube contained one of the alternative V primers together with the C primer. The first PCR cycle started with 2.5 minutes in a hot block at 95°C and then at 56°C (40 seconds) and 72°C (60 seconds). The ensuing 34 cycles consisted of 94°C (40 seconds), 56°C (40 seconds), and 72°C (60 seconds) in a Tetrad (MJ Research). PCR products were analyzed on a high-resolving polyacrylamide gel electrophoresis system in an ABI model 377 automatic DNA sequencer (Perkin-Elmer). Data were analyzed with the Genotyper 2.0 software program.

View this table: [in this window] [in a new window]   Table 2. PCR Primers for TCR and ß Chains

	Results

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ApoE-KO mice were divided into 2 groups that were either fed a high-cholesterol diet with 1.25% cholesterol or a western diet containing 0.15% cholesterol. This leads to different progression rates: whereas mice fed a western diet for 10 weeks exhibited fatty streaks, those fed the high-cholesterol diet for the same length of time had developed fibrofatty plaques.^{33 34} The latter type of lesions were observed in mice maintained on the western diet for 18 weeks, whereas mice fed the high-cholesterol diet for 18 weeks exhibited large, advanced fibrofatty plaques (Figure 1). The different diet treatments result in different immune effector mechanisms,¹⁶ and the present design permitted us to evaluate whether the immune detector mechanisms also varied depending on serum cholesterol levels or disease progression rate.

View larger version (126K): [in this window] [in a new window]   Figure 1. CD4+ T cells in fibrofatty plaques from aortic root of apoE-/- mice. Immunohistochemical staining of lesions from mice fed the high-cholesterol diet for 10 weeks (EH10) or 18 weeks (EH18) as well as mice fed the western diet for 18 weeks (EW18) is shown. The frames in the top panels are shown in the bottom panels at higher magnification. Magnification x50 (top panels) and x200 (bottom panels). The arrows point at sites in lesions that stain positive for CD4+ T cells. A calcification is marked with an asterisk.

An immunohistochemical analysis of lesions from the different groups of mice confirmed the abundance of CD4⁺ T cells in all types of lesions (Figure 1).

Atherosclerotic lesions were microdissected from the aortic root of each mouse. The sample from each mouse (Table 1) was treated individually (except samples 8 to 10; see Methods). The mRNA was isolated individually and used for RT-PCR analysis (see below). For comparison, mRNA from lymph node tissue was also analyzed. The TCR V region repertoire was analyzed by using 5' primers specific for the different V domains in combination with a common 3' primer from the C domain. This analysis provides information about the usage of different V domains by T cells in lesions. However, immune specificity is determined not only by V domain usage but also by nucleotide transferase activity associated with recombination events during T-cell differentiation. This activity adds or removes nucleotides at the various V-J, Vß-Dß, and Dß-Jß junctions in the maturing TCR genes and results in variations in the length of the mature TCR and TCRß genes. Together, V domain usage and junctional variability determine the conformation of the complementarity-determining region 3 (CDR3 region) of the TCR, which in turn determines the binding specificity of the antigen receptor.^{35 36} To assess junctional diversity as well as V domain usage, PCR was followed by fragment length analysis on high-resolution polyacrylamide gels in a DNA sequencer. The spectrum of size differences across the CDR3 region is known as the spectratype. Because the relative intensity of a given size peak is proportional to the amount of mRNA molecules in the starting material, an increase in height and area of a particular peak signals expansion of T-cell clones.¹⁷

When V and Vß profiles were run on lymph nodes, each TCR type (except V9 and Vß12) showed a gaussian distribution (Figures 2 and 3). Each peak within one V region profile differed by 3 bp, corresponding to peptide differences of 1 amino acid. Five to 7 different peaks were found for each TCR fragment, corresponding to a variation in length of 5 to 7 amino acids within the CDR3 region.

View larger version (41K): [in this window] [in a new window]   Figure 2. Spectratype profiles of the CDR3 length diversity in the V chain mRNA of TCR in atherosclerotic plaques. mRNA was extracted from plaques or lymph nodes (control tissue), and RT-PCR analysis of the CDR3 region in the TCR was performed as described in Methods. The PCR reaction was run with a fluorescently labeled common C-region primer and V-region–specific primers for each V. PCR products were analyzed on high-resolving PAGE in an automatic sequencer. The y axis shows fluorescence intensity; the x axis, CDR3 size. The boxed numbers show the length in base pairs of the PCR products in the corresponding peak. Each row contains all PCR products derived from one sample with use of the complete panel of V primers. All rows show the analysis from individual apoE-/- mice (m2, m3, etc), except for m8 to 10, for which samples from 3 mice were pooled. Sample number (m2 to m13), time on diet in weeks (w), and type of diet (either high-cholesterol diet [HCD] or western diet [WD]) are indicated. Top row, Lymph nodes of apoE-/- mice fed HCD for 10 weeks show gaussian distribution of CDR3 profiles corresponding to a polyclonal T-cell population. Subsequent rows, Plaques from apoE-/- mice have strongly skewed repertoires for the V TCR, indicating monoclonal or oligoclonal T-cell populations.

View larger version (46K): [in this window] [in a new window]   Figure 3. Spectratype profiles of the CDR3 length diversity in the Vß chain of the TCR in T cells infiltrating the atherosclerotic plaque. The analysis was performed as in Figure 2 and on the same samples; eg, sample m2 is from the same mouse in both V and Vß analyses. Top row, Lymph nodes of apoE-/- mice fed HCD for 10 weeks show gaussian distribution of CDR3 profiles corresponding to a polyclonal T-cell population. Subsequent rows, Plaques of apoE-/- mice exhibit strongly skewed repertoires for the Vß TCR, indicating monoclonal or oligoclonal T-cell population.

The TCR analysis of plaque tissue gave a totally different result, as shown in Figures 2 and 3 and in Table 3. First, only a limited set of V domains was represented in each of the 10 samples. The number of V domains expressed in each aorta varied between 1 and 8, with an average of 5.2 V and 5.6 Vß domains. Second, the detected TCR fragments exhibited strongly skewed profiles and never showed the gaussian distribution found in lymph nodes. In several samples, only one peak was visible, implying a monotypic expansion of T cells (eg, Figure 3, row m2, Vß6).

View this table: [in this window] [in a new window]   Table 3. Frequency of V Regions Found in TCR-Infiltrating Plaque

Vß6 was expressed as a dominant peak in all aortic samples (Figure 3 and Table 3). In 4 of them, a single peak of TCR Vß6 was observed, indicating a monoclonal T-cell population within these plaques (eg, Figure 3, rows m2 and m7). In the 6 remaining samples, 2 expanded peaks were identified (eg, Figure 3, rows m5 and m6). The second most common Vß domains, Vß5.2 and Vß16, were expressed in 5 of the 10 samples.

In the V analysis, the most frequent V domain was 34S, which was found in 9 of 10 samples (Table 3). As for Vß6, the profile was skewed, and one peak was dominant. The second most common V domain expressed was V9, which was also present as a single peak. However, it occurred as a single peak also in the lymph nodes. This transcript is probably derived from a rearranged pseudogene.²²

When comparing the mice fed a western diet with those maintained on the high-cholesterol diet, we found no differences in TCR profiles. In contrast, the stage of lesions appeared to influence the TCR patterns: more advanced lesions exhibited a more restricted TCR heterogeneity than did earlier stages of disease. This was particularly evident for Vß domains; compare samples m2 to m4 in Figure 3, which represent advanced fibrofatty lesions, with samples m11 to m13, which are derived from early fibrofatty plaques.

	Discussion

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A cellular immune response to a specific antigen induces proliferation of antigen-specific T cells, and this leads to expansion of T-cell clones carrying one unique TCR. Because T cells are important components of the atherosclerotic lesion at all stages and may recognize antigens present in the lesions, it is important to clarify whether clonal expansions of antigen-specific T cells occur in atherosclerosis.^{1 3} Previous studies of complicated human carotid lesions have failed to provide evidence for such expansions,^{13 14} but the situation may be entirely different in other phases of atherosclerosis. The development of murine models of the disease permits a dissection of all the phases of the disease with the use of clonotypic T-cell markers. The use of the recently developed spectratyping RT-PCR technique reveals the V and Vß gene usage together with the unique length of the CDR3 region in each T cell and serves as a clonotypic indicator.¹⁷ Because the mouse models have defined genotypes and not the same degree of complexity in the major histocompatibility complex (MHC) gene cluster as the human patients show in their HLA, this will be reflected in the TCR usage. In particular, the V domain usage will be less diverse, because all the mice in the present study carry the same MHC genes. Finally, the sensitivity of spectratyping allows an analysis of the antigen-binding domain of the TCR at a level that was not possible before when only Southern blot analysis¹⁴ or standard PCR¹³ was available.

With this approach of spectratyping RT-PCR of the TCR, we now demonstrate that the T-cell population of atherosclerotic lesions is highly skewed compared with the gaussian distribution found in lymph nodes. Not only is the representation of V domains much more limited in the lesions, but the CDR3 variability is also much more restricted. Interestingly, TCR heterogeneity was more reduced in the more mature plaques compared with earlier ones. This could imply that a heterogeneous population of T cells is initially recruited to the forming lesion by antigenically nonspecific mechanisms, followed by a selective expansion of T cells carrying specific reactivity to local antigens. The comparison with very advanced human lesions suggests that such a selective expansion is followed by extended chronic inflammation, leading to adhesion and immigration of heterogeneous T cells and a dilution of the specific population. This possibility should be tested when the precise specificities of the TCR represented in the human lesions are known.

One TCR V domain was represented in all lesions: Vß6. Only 1 or 2 Vß6 peaks were found in most samples, and the dominant peaks were 179 to 182 bp and thus differed by only 1 codon. It seems likely that this expansion of TCR Vß6 was caused by proliferation of antigen-specific T cells carrying this TCR. This expansion could theoretically arise either from an expanded population of activated Vß6⁺ T cells in the blood or by local proliferation of Vß6⁺ T cells in the plaque. Because no expansion of Vß6⁺ T cells was observed in lymph nodes, it is less probable that a systemically expanded Vß6 population was recruited to the lesion. Instead, the most likely explanation for the selective expansion of Vß6 mRNA in lesions is that Vß6⁺ T cells undergo local clonal proliferation in the lesion itself. This, in turn, suggests that plaque components are presented as antigens to local T cells. We have previously shown that CD4⁺ T cells of human lesions recognize oxidized LDL, and we have recently observed that murine T-cell hybridomas reactive with oxidation-induced LDL epitopes express Vß6 (A. Nicoletti, G. Paulsson, G.K. Hansson, unpublished observation, 1999). Together, these results suggest that TCR Vß6 is used in the recognition of oxidized LDL in atherosclerotic lesions, leading to clonal expansion of CD4⁺ T cells carrying this type of TCR.

In contrast to the dominance of Vß6, no single V domain was expressed in any lesion sample. Although TCR V34S was present in 9 of 10 lesion samples, it was missing in 1 of 3 early fibrofatty plaques of the apoE-KO mice fed a western diet (Figure 2, m7). Therefore, V expression was more heterogeneous than Vß expression, possibly reflecting a promiscuous usage of V domains in the generation of antigen-specific TCR–recognizing plaque antigens. This interpretation is in line with recent observations that each T-cell epitope can be recognized by many different TCRs and that each clonotypic TCR can recognize many different epitopes.^{37 38}

In spite of the relative heterogeneity of V expression, the selective expression of T cells carrying Vß6 suggests that such cells are important in the pathogenesis of atherosclerosis. Autoimmune diseases such as experimental autoimmune encephalomyelitis have been reversed by treatment with monoclonal antibodies specific for the TCR-Vß type carried by disease-mediating T cells.³⁹ It will now be interesting to determine whether atherosclerosis can also be controlled by such reagents.

	Acknowledgments

 
This study was supported by grants from the Swedish Medical Research Council (project No. 6816), the Heart-Lung Foundation, and the Hedlund, Johnson, Tore Nilsson, Nanna Svartz, King Gustaf V, and Gamla Tjänarinnor funds. E. Törnquist was supported by a fellowship from the National Network for Cardiovascular Research. We thank Ingrid Törnberg for excellent technical assistance.

Received May 5, 1999; accepted June 10, 1999.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages, and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis. 1986;6:131–138.[Abstract/Free Full Text]
2. Libby P, Hansson GK. Involvement of the immune system in human atherogenesis: current knowledge and unanswered questions. Lab Invest. 1991;64:5–15.[Medline] [Order article via Infotrieve]
3. Zhou X, Stemme S, Hansson GK. Evidence for a local immune response in atherosclerosis: CD4+ T cells infiltrate lesions of apo E-deficient mice. Am J Pathol.. 1996;149:359–366.[Abstract]
4. Hansson GK. Cell-mediated immunity in atherosclerosis. Curr Opin Lipidol.. 1997;8:301–311.[Medline] [Order article via Infotrieve]
5. Hansson GK, Stemme V, Yokota T; Remick D, Friedland J, eds. Cytokines and the Cardiovascular System. Philadelphia, Pa: Lippincott-Raven Publishers; 1997:507–517.
6. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995;91:2844–2850.[Free Full Text]
7. Liuzzo G, Biasucci LM, Rebuzzi AG, Gallimore R, Caligiuri G, Lanza GA, Quaranta G, Monaco C, Pepys MB, Maseri A. Plasma protein acute-phase response in unstable angina is not induced by ischemic injury. Circulation. 1996;94:2373–2380.[Abstract/Free Full Text]
8. Caligiuri G, Liuzzo G, Biasucci LM, Maseri A. Immune system activation follows inflammation in unstable angina: pathogenetic implications. J Am Coll Cardiol. 1998;32:1295–1304.[Abstract/Free Full Text]
9. Ylä-Herttuala S, Palinski W, Butler SW, Picard S, Steinberg D, Witztum JL. Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler Thromb. 1994;14:32–40.[Abstract/Free Full Text]
10. Xu Q, Kleindienst R, Waitz W, Dietrich H, Wick G. Increased expression of heat shock protein 65 coincides with a population of infiltrating T lymphocytes in atherosclerotic lesions of rabbits specifically responding to heat shock protein 65. J Clin Invest.. 1993;91:2693–702.
11. Kuo CC, Shor A, Campbell LA, Fukushi H, Patton DL, Grayston JT. Demonstration of Chlamydia pneumoniae in atherosclerotic lesions of coronary arteries. J Infect Dis. 1993;167:841–849.[Medline] [Order article via Infotrieve]
12. Stemme S, Holm J, Hansson GK. T lymphocytes in human atherosclerotic plaques are memory cells expressing CD45RO and the integrin VLA-1. Arterioscler Thromb.. 1992;12:206–211.[Abstract/Free Full Text]
13. Swanson SJ, Rosenzweig A, Seidman JG, Libby P. Diversity of T-cell antigen receptor V beta gene utilization in advanced human atheroma. Arterioscler Thromb. 1994;14:1210–1214.[Abstract/Free Full Text]
14. Stemme S, Rymo L, Hansson GK. Polyclonal origin of T lymphocytes in human atherosclerotic plaques. Lab Invest. 1991;65:654–660.[Medline] [Order article via Infotrieve]
15. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A.. 1992;89:4471–4475.[Abstract/Free Full Text]
16. Zhou X, Paulsson G, Stemme S, Hansson GK. Hypercholesterolemia is associated with a Th1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout mice. J Clin Invest. 1998;101:1717–1725.[Medline] [Order article via Infotrieve]
17. Pannetier C, Cochet M, Darche S, Casrouge A, Zoller M, Kourilsky P. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proc Natl Acad Sci U S A. 1993;90:4319–4323.[Abstract/Free Full Text]
18. Patten P, Yokota T, Rothbard J, Chien Y, Arai K, Davis MM. Structure, expression and divergence of T-cell receptor beta-chain variable regions. Nature. 1984;312:40–46.[Medline] [Order article via Infotrieve]
19. Chien YH, Gascoigne RJ, Kavaler J, Lee NE, Davis MM. Somatic recombination in a murine T-cell receptor gene. Nature. 1984;309:322–326.[Medline] [Order article via Infotrieve]
20. Chien Y, Becker DM, Lindsten T, Okamura M, Cohen DI, Davis MM. A third type of murine T-cell receptor gene. Nature. 1984;312:31–35.[Medline] [Order article via Infotrieve]
21. Gascoigne NR, Chien Y, Becker DM, Kavaler J, Davis MM. Genomic organization and sequence of T-cell receptor beta-chain constant- and joining-region genes. Nature. 1984;310:387–391.[Medline] [Order article via Infotrieve]
22. Arden B, Klotz JL, Siu G, Hood LE. Diversity and structure of genes of the alpha family of mouse T-cell antigen receptor. Nature. 1985;316:783–787.[Medline] [Order article via Infotrieve]
23. Barth RK, Kim BS, Lan NC, Hunkapiller T, Sobieck N, Winoto A, Gershenfeld H, Okada C, Hansburg D, Weissman IL, et al. The murine T-cell receptor uses a limited repertoire of expressed V beta gene segments. Nature. 1985;316:517–523.[Medline] [Order article via Infotrieve]
24. Behlke MA, Loh DY. Alternative splicing of murine T-cell receptor beta-chain transcripts. Nature. 1986;322:379–382.[Medline] [Order article via Infotrieve]
25. Behlke MA, Chou HS, Huppi K, Loh DY. Murine T-cell receptor mutants with deletions of beta-chain variable region genes. Proc Natl Acad Sci U S A. 1986;83:767–771.[Abstract/Free Full Text]
26. Chou HS, Behlke MA, Godambe SA, Russell JH, Brooks CG, Loh DY. T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments. EMBO J. 1986;5:2149–2155.[Medline] [Order article via Infotrieve]
27. Tillinghast JP, Behlke MA, Loh DY. Structure and diversity of the human T-cell receptor beta-chain variable region genes. Science. 1986;233:879–883.[Abstract/Free Full Text]
28. Chou HS, Nelson CA, Godambe SA, Chaplin DD, Loh DY. Germline organization of the murine T cell receptor beta-chain genes. Science. 1987;238:545–548.[Abstract/Free Full Text]
29. Chou HS, Anderson SJ, Louie MC, Godambe SA, Pozzi MR, Behlke MA, Huppi K, Loh DY. Tandem linkage and unusual RNA splicing of the T-cell receptor beta-chain variable-region genes. Proc Natl Acad Sci U S A. 1987;84:1992–1996.[Abstract/Free Full Text]
30. Kappler JW, Wade T, White J, Kushnir E, Blackman M, Bill J, Roehm N, Marrack P. A T cell receptor V beta segment that imparts reactivity to a class II major histocompatibility complex product. Cell. 1987;49:263–271.[Medline] [Order article via Infotrieve]
31. Koide Y, Kaidoh T, Yanagawa T, Yoshida TO. A comparative study on T cell receptor V beta gene usages: spleen cells from the non-obese diabetic (NOD) mouse and its non-diabetic sister strain, the ILI mouse, and infiltrating T cells into pancreata of NOD mice. Microbiol Immunol. 1993;37:653–659.[Medline] [Order article via Infotrieve]
32. Casanova JL, Romero P, Widmann C, Kourilsky P, Maryanski JL. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J Exp Med. 1991;174:1371–1383.[Abstract/Free Full Text]
33. Reddick RL, Zhang SH, Maeda N. Atherosclerosis in mice lacking apo E: evaluation of lesional development and progression [published erratum appears in Arterioscler Thromb. 1994;14:839]. Arterioscler Thromb. 1994;14:141–147.
34. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994;14:133–140.[Abstract/Free Full Text]
35. Chothia C, Boswell DR, Lesk AM. The outline structure of the T-cell alpha beta receptor. EMBO J. 1988;7:3745–3755.[Medline] [Order article via Infotrieve]
36. Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cell recognition [published erratum appears in Nature. 1988;20;335:744]. Nature.. 1988;334:395–402.[Medline] [Order article via Infotrieve]
37. Breiteneder H, Friedl HR, Ebner C, Schenk S, Fischer G, Kraft D, Scheiner O. Sequence comparisons of the CDR3 hyper-variable loops of human T cell receptors specific for three major T cell epitopes of the birch pollen allergen Bet v 1. Mol Immunol.. 1996;33:1039–1048.[Medline] [Order article via Infotrieve]
38. Gapin L, Fukui Y, Kanellopoulos J, Sano T, Casrouge A, Malier V, Beaudoing E, Gautheret D, Claverie JM, Sasazuki T, Kourilsky P. Quantitative analysis of the T cell repertoire selected by a single peptide-major histocompatibility complex. J Exp Med. 1998;187:1871–1883.[Abstract/Free Full Text]
39. Acha-Orbea H, Mitchell DJ, Timmermann L, Wraith DC, Tausch GS, Waldor MK, Zamvil SS, McDevitt HO, Steinman L. Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell. 1988;54:263–273.[Medline] [Order article via Infotrieve]
40. Breslow JL. Mouse models of atherosclerosis. Science. 1996;272:685–688.[Abstract]

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				H. Bagavant, U. S. Deshmukh, H. Wang, T. Ly, and S. M. Fu Role for Nephritogenic T Cells in Lupus Glomerulonephritis: Progression to Renal Failure Is Accompanied by T Cell Activation and Expansion in Regional Lymph Nodes J. Immunol., December 1, 2006; 177(11): 8258 - 8265. [Abstract] [Full Text] [PDF]

				A.-K. L. Robertson and G. K Hansson T Cells in Atherogenesis: For Better or For Worse? Arterioscler. Thromb. Vasc. Biol., November 1, 2006; 26(11): 2421 - 2432. [Abstract] [Full Text] [PDF]

				E. Galkina, A. Kadl, J. Sanders, D. Varughese, I. J. Sarembock, and K. Ley Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent J. Exp. Med., May 15, 2006; 203(5): 1273 - 1282. [Abstract] [Full Text] [PDF]

				J. Khallou-Laschet, G. Caligiuri, E. Groyer, E. Tupin, A.-T. Gaston, B. Poirier, M. Kronenberg, J. L. Cohen, D. Klatzmann, S. V. Kaveri, et al. The Proatherogenic Role of T Cells Requires Cell Division and Is Dependent on the Stage of the Disease Arterioscler. Thromb. Vasc. Biol., February 1, 2006; 26(2): 353 - 358. [Abstract] [Full Text] [PDF]

				V. C. Mehra, V. S. Ramgolam, and J. R. Bender Cytokines and cardiovascular disease J. Leukoc. Biol., October 1, 2005; 78(4): 805 - 818. [Abstract] [Full Text] [PDF]

				X. Zhou, A.-K. L. Robertson, M. Rudling, P. Parini, and G. K. Hansson Lesion Development and Response to Immunization Reveal a Complex Role for CD4 in Atherosclerosis Circ. Res., March 4, 2005; 96(4): 427 - 434. [Abstract] [Full Text] [PDF]

				T. J. DeGraba Immunogenetic Susceptibility of Atherosclerotic Stroke: Implications on Current and Future Treatment of Vascular Inflammation Stroke, November 1, 2004; 35(11_suppl_1): 2712 - 2719. [Abstract] [Full Text] [PDF]

				E. Tupin, A. Nicoletti, R. Elhage, M. Rudling, H.-G. Ljunggren, G. K. Hansson, and G. P. Berne CD1d-dependent Activation of NKT Cells Aggravates Atherosclerosis J. Exp. Med., February 2, 2004; 199(3): 417 - 422. [Abstract] [Full Text] [PDF]

				O. J de Boer, A. E Becker, and A. C van der Wal T lymphocytes in atherogenesis--functional aspects and antigenic repertoire Cardiovasc Res, October 15, 2003; 60(1): 78 - 86. [Full Text] [PDF]

				G. K. Hansson, P. Libby, U. Schonbeck, and Z.-Q. Yan Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis Circ. Res., August 23, 2002; 91(4): 281 - 291. [Abstract] [Full Text] [PDF]

				S. A. Schreyer, C. M. Vick, and R. C. LeBoeuf Loss of Lymphotoxin-alpha but Not Tumor Necrosis Factor-alpha Reduces Atherosclerosis in Mice J. Biol. Chem., March 29, 2002; 277(14): 12364 - 12368. [Abstract] [Full Text] [PDF]

				Q. Zhang, X. Zeng, J. Guo, and X. Wang Oxidant stress mechanism of homocysteine potentiating Con A-induced proliferation in murine splenic T lymphocytes Cardiovasc Res, March 1, 2002; 53(4): 1035 - 1042. [Abstract] [Full Text] [PDF]

				G. K. Hansson Immune Mechanisms in Atherosclerosis Arterioscler. Thromb. Vasc. Biol., December 1, 2001; 21(12): 1876 - 1890. [Abstract] [Full Text] [PDF]

				Z. G. Nadareishvili, D. E. Koziol, B. Szekely, C. Ruetzler, R. LaBiche, R. McCarron, T. J. DeGraba, and S. Jander Increased CD8+ T Cells Associated With Chlamydia pneumoniae in Symptomatic Carotid Plaque Editorial Comment Stroke, September 1, 2001; 32(9): 1966 - 1972. [Abstract] [Full Text] [PDF]

				S. Stemme Plaque T-Cell Activity : Not So Specific? Arterioscler. Thromb. Vasc. Biol., July 1, 2001; 21(7): 1099 - 1101. [Full Text] [PDF]

				C. A. Reardon, L. Blachowicz, T. White, V. Cabana, Y. Wang, J. Lukens, J. Bluestone, and G. S. Getz Effect of Immune Deficiency on Lipoproteins and Atherosclerosis in Male Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2001; 21(6): 1011 - 1016. [Abstract] [Full Text] [PDF]

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