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* Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
Division of Health Sciences and Technology, Massachusetts Institute of Technology, Harvard Medical School, Boston, MA 02115;
Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129;
Laboratory Medicine Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and
¶ Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, MA 02115
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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T-bet, a member of the T-box family of transcription factors, is a regulator required for driving differentiation of naive CD4+ T cells toward the Th1 phenotype. T-bet is also expressed in CD8+ T cells, but its role in the differentiation of CTL effectors is less well-characterized. In vitro analysis has shown that T-bet is required for Ag-driven differentiation of cytotoxic activity of CD8+ T cells, and T-bet regulates the profile of cytokines secreted by CD8+ effector cells (6). Mice lacking T-bet are impaired in their ability to mount protective CD8+ T cell responses to lymphocytic choriomeningitis virus. Furthermore, in a transgenic model of lymphocytic choriomeningitis virus-induced diabetes that is dependent on CD8+ T cells, T-bet deficiency protects against development of disease (7) through a mechanism that is incompletely understood.
An important component of the differentiation of effector T cells is the expression of molecules that are required for efficient migration into inflamed tissues. Studies of Th cell subset differentiation indicate that conditions that favor Th1 differentiation, such as the presence of IL-12 during initial activation of naive CD4+ T cells, also favor the expression of functional selectin ligands (8, 9, 10, 11, 12) and certain chemokine receptors, especially CXCR3 and CCR5 (13). Blockage of CXCR3 prevents disease progression in a model of experimental autoimmune myocarditis (14). It was recently demonstrated that T-bet is required for the expression of CXCR3 and functional P-selectin ligands on CD4+ Th cells in vitro (15). Subsequently, the CXCR3 promoter was identified as a direct binding target of T-bet in lymphocytes, but the functional consequences of this binding in the CD8+ T cell subset are not clear (16).
To explore the possible role of T-bet in the differentiation of pathogenic CD8+ effectors in cardiac disease, we used a transgenic mouse model of CD8+ T cell-mediated myocarditis (cMy-mOVA) (17, 18). IL-12 plays a critical role in the differentiation of effector CD8+ T cells that cause disease in cMy-mOVA mice (17). This model facilitates the analysis of migration and effector function of a monospecific population of CD8+ T cells that recognize an Ag expressed exclusively in the heart. In this way, it is advantageous to a coxsackievirus model which does not distinguish between direct viral damage vs immune-mediated injury, and also to experimental autoimmune myocarditis, which relies on immunization with exogenous protein Ags that are preferentially processed by the class II MHC pathway and may not efficiently induce CD8+ T cell responses. Further, the cMy-mOVA model allows specific genetic defects to be mapped exclusively to the transferred T cells.
In the present study, we examined the influence of T-bet deficiency on the ability of CD8+ effector T cells to traffic into the heart, and separately, to cause damage there. We found that the pathogenicity of cardiac-Ag-specific CD8+ T cells was markedly reduced in the absence of T-bet, and this phenotype was due, in significant part, to diminished CXCR3-dependent migration of the T cells into the heart. Nonetheless, reconstitution of migration via retroviral-mediated expression of CXCR3 was not sufficient to restore cardiac damage by T-bet-deficient CTL. This likely reflects the reduced cytotoxicity and enhanced extrinsic and intrinsic regulation of these cells. Our results clearly demonstrate that T-bet regulates multiple components of the effector phenotype of CD8+ T cells, including chemokine receptor and cytokine expression, cytotoxicity, and susceptibility to regulatory mechanisms that normally control T cell-mediated immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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All mice used in the current study were bred in the pathogen-free facility at the Warren Alpert Building (Harvard Medical School, Boston, MA), in accordance with the guidelines of the Committee of Animal Research at the Harvard Medical School and the National Institutes of Health Animal Research Guidelines. The cMy-mOVA-transgenic mouse line that expresses OVA in the heart (17) was carried on both C57BL/6-Thy 1.2 (CD90.2) and Thy 1.1 (CD90.1) backgrounds, and all experimental animals were heterozygous for the OVA transgene. The OT-I TCR-transgenic mouse strain (19) was provided by W. R. Heath and F. Carbone (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) and was maintained on a C57BL/6-Thy 1.2 (CD90.2) background. The OT-I TCR is expressed on CD8+ T cells and is specific for the OVA peptide p.257–264 (SIINFEKL) bound to the class I MHC molecule H2-Kb (20). OT-I TCR-transgenic mice lacking T-bet were derived as previously described (6). CXCR3-deficient mice backcrossed more than six times on a C57BL/6 background (21) were provided by C. Gerard (Children’s Hospital, Boston MA), and were cross-bred with OT-I mice to generate a CXCR3–/–OT-I line. C57BL/6 mice were purchased from The Jackson Laboratory.
T cell preparations
Cells were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 2 mM/L sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM/L HEPES (Invitrogen Life Technologies). Cell suspensions were prepared from spleen and lymph node of OT-I T-bet+/+ or OT-I T-bet–/– TCR-transgenic mice, and naive OT-I cells were isolated from the suspensions by CD8 magnetic beads (Miltenyi Biotec), as previously described (17, 18). The naive T cells were stimulated in vitro with mitomycin-C (Sigma-Aldrich) treated C57BL/6 spleen cell suspensions (APCs) at a T cell:APC ratio of 1:10, and OVA peptide Ag (SIINFEKL) was added at a final concentration of 1 µM/L. These cultures were supplemented with 2 µg/ml anti-CD28 (BD Pharmingen), 50 U/ml recombinant mouse IL-2 (R&D Systems), and 10 ng/ml recombinant mouse IL-12 (R&D Systems). The cultures were placed in 75 cm2 flasks and incubated at 37°C, 5% CO2. After 3 days of stimulation, all cultures were diluted 1/1 with fresh medium containing 40 U/ml IL-2 (R&D Systems) and OT-I effectors were harvested for use at day 5 or 6.
Retroviral transduction
Human CXCR3 was subcloned into the retroviral expression plasmid pMigR, a gift of Dr. G. Nolan (Stanford University, Stanford, CA), between the EcoR1 and XhoI restriction sites. This plasmid encodes an internal ribosome entry site followed by the gene for enhanced GFP (EGFP),3 allowing for the identification of transduced cells by analyzing for EGFP expression. The resulting plasmid was named pCXCR3-MigR. pCXCR3-MigR and a plasmid encoding the vesicular stomatitis virus glycoprotein were transfected into HEK293 cells using Fugene 6 (Roche) and 72 h later the virus-containing supernatant was harvested. On day 3 following the isolation and stimulation, 5 x 106 OT-1 CD8+ T cells were incubated in 25 ml of supernatant obtained from HEK293 cells transfected with pCXCR3-MigR or control virus at a titer of 
105 infectious particles per milliliter. Polybrene was added to a final concentration of 7.5 µg/ml and the cells were centrifuged at 2000 rpm for 90 min. The retrovirus containing supernatant was removed and replaced with RPMI 1640 containing 10% FBS (Sigma-Aldrich). Seventy-two hours following transduction, the percentage of human CXCR3-expressing cells was determined by staining the cells with anti-CXCR3 (R&D Systems) and analyzing by flow cytometry. A >95% correlation of human CXCR3 and EGFP expression was consistently obtained (data not shown).
Adoptive transfer
In vitro-activated OT-I effector cells were removed form tissue culture, washed, and resuspended in PBS and injected i.p. into cMy-mOVA mice, as previously described (17, 18). Cell doses transferred in this study ranged from 0.25 to 1.0 x 106 cells per mouse as indicated.
Processing of tissue
At designated times after adoptive transfer, usually 72–96 h, cMy-mOVA mice were anesthetized by i.p. injection of 2,2,2 tribromoethanol (0.5 mg/g). The chest cavity of each mouse was then opened, the inferior vena cava was nicked, and the left ventricle was perfused with cold PBS via a left ventricle apical puncture with an 18° syringe needle. After exsanguination, the heart was surgically removed, placed in ice-cold RPMI 1640 medium, and cut with a scalpel to yield three contiguous transverse biventricular sections. The basal section was frozen in Tissue-Tek OCT compound (Sekura Finetek) and stored at –80°C for subsequent immunohistochemical staining. The midportion was fixed with 10% phosphate-buffered formalin, embedded in paraffin, and used for preparation of H&E-stained sections. The apical portion was fixed in TRIzol reagent (Invitrogen Life Technologies) for subsequent RNA extraction. In a subset of animals from some experiments, coronal sections of the heart including atria and ventricles were prepared for routine histology.
Grading myocarditis
Myocarditis was graded by microscopic examination of H&E-stained sections in a blinded fashion by the same trained pathologist after examination of the entire area of three sections, using a 0–4 scale as follows: 0, no inflammation; 1, one to five distinct inflammatory foci with total involvement of 5% or less of the cross-sectional area; 2, more than five distinct inflammatory foci, or involvement of >5% but <20% of the cross-sectional area; 3, diffuse inflammation involving over 20–50% of the area; 4, diffuse inflammation involving >50% of the area.
Immunohistochemistry
Immunohistochemistry was performed as previously described (22). Briefly, acetone-fixed cryostat sections of heart were blocked with 1% BSA in PBS. All Abs were purchased from BD Pharmingen, unless specified otherwise. Sections were incubated with 10 µg/ml unlabeled primary rat anti-mouse Ig (specific for CD4, CD8, or PD-L1), followed by PBS wash, and then biotinylated goat anti-rat Ig, 1/200 (Jackson ImmunoResearch Laboratories). For Thy 1.2 staining, sections were incubated with biotinylated anti-CD90.2 Ab (BD Pharmingen). All sections were then blocked with 0.3% hydroperoxide/PBS at room temperature, and incubated with HRP-avidin-biotin complex solutions at a 1/100 dilution (Vector Laboratories). Specific Ab binding was detected with 3-amino-9-ethylcarbazole (Vector Laboratories) and counterstained with Gill’s number 2 hematoxylin solution (Polysciences).
In vitro analysis of T cell functional phenotype
OT-I T-bet+/+ or OT-I T-bet–/– effector cells were removed from primary activation cultures at day 5, washed, and resuspended in RPMI 1640 medium, and used for functional assays. Cytotoxic activity of effector OT-I cells was measured by coculturing the T cells with [51Cr]sodium chromate loaded H-2Kb-expressing EL4 target cells (American Type Culture Collection) at various E:T cell ratios. Target cells were pulsed with SIINFEKL, and released 51Cr in supernatants was detected by gamma counting, as described (17). Cytokine secretion by effector T cells was stimulated by culturing 2 x 104 effector T cells with 2 x 105 mitomycin C (Sigma-Aldrich) treated C57BL/6 spleen cells and SIINFEKL (1 µg/ml) in 200-µl microtiter wells, or by culturing 2 x 104 T cells in microwells precoated with anti-CD3
(145:2C11; BD Pharmingen). Culture supernatants were harvested at 48 h and cytokines were detected by cytometric-bead assay (BD Biosciences).
Serum troponin I determination
Blood was collected from mice at time of sacrifice, and serum fractions were isolated and stored frozen before cardiac troponin I (TnI) determinations. TnI was measured using the ADVIA Centaur cTnI assay (Bayer), as previously described (23).
Flow cytometry analyses
All cell preparations were washed twice in staining buffer (Dulbecco’s PBS with 1% BSA). For phenotypic analysis of surface markers, 0.5 x 106 cells were suspended in 100 µl of staining buffer containing 1 µg of each specific Ab, and incubated on ice for 20 min followed by washing and fixation with 0.5% paraformaldehyde. Stained cell preparations were then analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD Biosciences). Fluorochrome-conjugated mouse-specific Abs purchased from BD Pharmingen unless specified otherwise, used for flow cytometry, were as follows: CD8 (clone 53-6.7); CD90.1/Thy 1.1 (clone OX-7); CD90.2/Thy 1.2 (clone 53.21); CD44 (clone IM7); CD25 (IL-2R 
-chain) (clone 7D4); CD62L (Mel-14); CD45Rb (clone 16A); V
5 (clone MR9-4); V2 (clone B20.1); CD69 (H12F3); CXCR3 (R&D Systems; clone 220803). In addition, we used anti-human CXCR3 (clone 1C6/CXCR3) from BD Pharmingen.
Real-time RT-PCR analysis
Quantitative real-time analysis of gene expression was performed on RNA samples extracted from cultured T cells or myocardium, as previously described (17). CD8+ spleen cells stimulated with anti-CD3 were analyzed for cytokine production by TaqMan real-time PCR (RT-PCR). Total RNA was isolated from 5 x 106 million purified T cells by TRIzol (Invitrogen Life Technologies). Residual traces of DNA were eliminated by DNase I (Invitrogen Life Technologies). Total RNA was quantified by absorbance at 260 nm and used as templates for reverse transcription of first-strand cDNA using the ThermoScript RT-PCR system and random hexamer primers (Invitrogen Life Technologies) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed in triplicates with SYBR Green PCR mix (Bio-Rad). All real-time reactions were conducted on the iCycler iQ Real-Time PCR Detection System (Bio-Rad) and analysis was done with the accompanying software. The presence of single amplicons resulting from real-time RT-PCR was verified by dissociation curve analysis. Levels of specific gene expression in the samples are presented relative to endogenous levels of -actin housekeeping gene expression in the same sample to normalize for mRNA differences between samples. The sequence of the forward and reverse primers, respectively, were: -actin, 5'-TCCTTCGTTGCCGGTCCA-3' and 5'-ACCAGCGCAGCGATATCGTC-3'; T-bet, 5'-ACAACCCCTTTGCCAAAG-3' and 5'-TCCCCCAAGCAGTTGACAGT-3'; CCR7, 5'-CACGCTGAGATGCTCACTGG-3' and 5'-CCATCTGGGCCACTTGGA-3'; CXCR3, 5'- TGCTGTGCTACTGAGTCAGCG-3' and 5'- CTACAGCCAGGTGGAGCAGG-3'; CCR5, 5'-CCATGCAGGCAACAGAGACTC-3' and 5'-TCTCTCCAACAAAGGCATAGATGA-3'; CCR2, 5'-TCAACTTGGCCATCTCTGACC-3' and 5'-AGACCCACTCATTTGCAGCAT-3'; granzyme b, 5'-GCCTTCTTCCTCTCCTAGAGGTT-3' and 5'-CGGAAGGCCGCCTAGGT-3'; perforin, 5'-CAGGTCAGGCCAGCATAAGAG-3' and 5'-TGGTTGGTGACCTTTGAATCC-3'; FasL, 5'-CAGGTCAGGCCAGCATAAGAG-3' and 5'-GGCTATTTGCTTTTCAAAAGATGATAC-3'; IFN-inducible protein 10 (IP-10), 5'-GCCGTCATTTTCTGCCTCA-3' and 5'-CGTCCTTGCGAGAGGGATC-3'; monokine induced by IFN-
(Mig), 5'-AATGCACGATGCTCCTGCA and 5'-AGGTCTTTGAGGGATTTGTAGTGG-3'; IFN-inducible T cell chemoattractant (I-TAC), 5'-CAGGAAGGTCACAGCCATAGC-3' and 5'-TTTCTCGATCTCTGCCATTTTG-3'; RANTES, 5'- CAAGTGCTCCAATCTTGCAGTC-3' and 5'- CAAGTGCTCCAATCTTGCAGTC-3'; MCP-1, 5'- TGGCTCAGCCAGATGCAGT-3' and 5'- TTGGGATCATCTTGCTGGTG-3'; FoxP3, 5'-GGCCCTTCTCCAGGACAGA-3' and 5'-GCTGATCATGGCTGGGTTGT-3'; TGF-1, 5'-TGACGTCACTGGAGTTGTACGG and 5'-GGTTCATGTCATGGATGGTGC-3'; programmed death-ligand 1 (PD-L1), 5'-ATGATGTTTTCTACTGTACGTTTTGGA-3' and 5'-GTGAGTCCTGTTCTGTGGAGGAT-3'; IFN-, 5'-AACGCTACACACTGCATCTTGG-3' and 5'-GCCGTGGCAGTAACAGCC-3'; Fas, 5'-GCAAACCAGACTTCTACTGCG-3' and 5'-TTTGTATTGCTGGTTGCTGTG-3'; thymus inhibitor of apoptosis (TIAP), 5'-CTACCGAGAACGAGCCTGATT-3' and 5'-CTTTTTGCTTGTTGTTGGTCTCC-3'; caspase 8, 5'-TCAACTTCCTAGACTGCAACCG-3' and 5'-CTCAATTCCAACTCGCTCACTT-3'; IL-12R 2, 5'-CAAGCATTTGCATCGCTATCA-3' and 5'-AATGCCTTTTGCCGGAAGT-3'; IL-17, 5'-GCTCCAGAAGGCCCTCAGA and 5'-AGCTTTCCCTCCGCATTGA-3'.
Chemotaxis assays
Chemotaxis assays of OT-1 cells were performed in 96-well Neuroprobe chemotaxis chambers with 5 µM pore size polycarbonate membranes (Neuroprobe) as previously reported (24). A total of 31 µl of RPMI 1640 containing 1% BSA and chemokines were placed in the bottom chamber of the device per the manufacturer’s directions. A total of 25,000 cells were layered onto the top of the membrane in RPMI 1640 containing 1% BSA. The chambers were then incubated at 37°C for 3 h. Following the incubation period, the migrating cells were removed from the bottom chambers and placed into plastic tubes, fixed in 2% paraformaldehyde/PBS. A total of 20,000 FACS counting beads were added to each sample (Polysciences). To control for the quantification of the migrating cells, flow cytometry was performed until 10,000 counting beads were counted. Chemotactic indices were calculated by dividing the number of cells in the bottom wells at each concentration by the number of cells in the bottom wells with no chemokine added.
Statistical analysis
Statistical analyses were performed using the two-tailed Student t test. A value of p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The studies described focus on effector CD8+ T cells generated under well-defined conditions by in vitro Ag-specific activation of naive OT-I cells. There was no significant difference in the number of naive CD8+ T cells isolated from lymph nodes and spleen of T-bet+/+ and T-bet–/– OT-I mice. In vitro Ag stimulation of T cells from both types of mice resulted in robust proliferative responses, with at least as many T-bet–/– OT-I effector cells as control OT-I cells recovered after 5–6 days of culture. Peptide Ag restimulation of the T-bet–/– OT-I effector cells generated in primary cultures resulted in a greater proliferative response than restimulation of control OT-I cells, over a range of peptide doses (Fig. 1A) as well as upon restimulation with anti-CD3 (data not shown). Furthermore, activation markers were expressed at comparable or higher levels on the T-bet–/– effectors compared with wild-type controls (Fig. 1B). These data indicate that the T-bet–/– effectors that we use for adoptive transfer experiments described below are viable, activated, and capable of responding to Ag.
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The pathogenicity of T-bet–/– and control OT-I effectors was compared in the cMy-mOVA model of myocarditis. Adoptive transfer of 250,000 T-bet–/– effectors resulted in 0% mortality over 90 days compared with 50% mortality (all deaths occurring by 16 days) among recipients of the same number of control OT-I cells (Fig. 2A). Among recipients of 500,000 T-bet–/– OT-I effectors, there was 16% overall mortality through 90 days compared with 100% mortality (all deaths occurring by day 8) among recipients of the same number of control OT-I cells (Fig. 2A). To directly assess myocardial damage, we measured concentrations of serum TnI at day 5 after T cell transfer to cMy-mOVA recipients. The mean TnI levels after transfer of 250,000 control or T-bet–/– OT-I effectors were 317 ± 82 and 7 ± 2 ng/ml, respectively (Fig. 2B). The TnI levels in recipients of 500,000 control or T-bet–/– OT-I effectors were 418 ± 56 and 0 ± 0 ng/ml, respectively (Fig. 2B). Even among mice with sublethal disease, there was a significant difference in the degree of myocardial damage mediated by control or T-bet–/– effectors, as discernable by serum TnI concentrations. Histopathological grading of severity of inflammation in hearts removed 5 days post-OT-I cell transfer also revealed that T-bet–/– OT-I cells were markedly less pathogenic than control OT-I cells (Fig. 2C). Hearts from cMy-mOVA recipients of 250,000 control or T-bet–/– OT-I effectors had scores of 2.5 ± 0.28 and 0.25 ± 0.25, respectively. The scores for recipients of 500,000 control or T-bet–/– OT-I effectors were 4.0 ± 0.00 and 1.0 ± 0.41, respectively.
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, IFN-, MCP-1, IL-12, and IL-6 was detectable in cMy-mOVA mice 4–5 days after transfer of control OT-I effector cells, but not in recipients of T-bet–/– OT-I cells (Fig. 2D and data not shown). None of these cytokines is detectable by our assays in the sera of unmanipulated cMy-mOVA mice (data not shown). The comparative histomorphology of the hearts in cMy-mOVA recipients of control or T-bet–/– OT-I effector cells is shown in Fig. 3. In contrast to the diffuse inflammatory infiltrate and associated myocyte injury seen in ventricular and atrial walls in recipients of control T cells, there was comparatively minimal myocardial infiltrate in recipients of T-bet–/– T cells. Because the inflammatory infiltrate in this model of myocarditis includes a significant number of endogenous leukocytes in addition to the transferred OT-I cells (18), it is possible that comparable numbers of control and T-bet–/– OT-I effectors migrated into the hearts, and the differences in the resulting histological appearance and damage are related to impaired ability of the T-bet–/– T cells to cause damage and secondary inflammation. To address this possibility, we took advantage of allotypic differences in CD90 between T cell donor and recipient animals to specifically identify the transferred T cells in the cardiac tissue. As shown in Fig. 3, E and F, numerous transferred control OT-I cells, but few if any transferred T-bet–/– OT-I cells are detectable by immunohistochemistry in the cMy-mOVA hearts by day 5. These data are consistent with a migratory defect of T-bet–/– CD8+ T cells; alternatively, they may reflect impaired ability of the T-bet-deficient cells to survive or proliferate in the myocardium.
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T-bet-deficient CD4+ effectors are skewed toward a Th2 phenotype (25). Previous studies with T-bet–/– OT-I CD8+ effectors indicate they have an altered cytokine secretion profile compared with control OT-I cells (6). It was therefore possible that the T-bet–/– CD8+ effectors we generated in this study also had a type-2 phenotype, which might alter their pathogenicity. One important difference between the current study and previous studies of T-bet-deficient CD8+ T cell effectors is that in the current study, IL-12 was included in the primary stimulation of naive T cells, a treatment which is required for generating effectors pathogenic to cMy-mOVA mice. Interestingly, measurements of cytokines secreted by T-bet–/– or control OT-I cells after in vitro restimulation with OVA peptide and APCs resemble a type-1 phenotype (Fig. 4A). T-bet–/– cells secreted significantly more IL-2 than control cells, which correlates with the higher proliferative activity of the T-bet–/– cells (see Fig. 1). We detected no IL-4 or IL-5 secretion by Ag-restimulated T-bet–/– control OT-I cells (data not shown). In contrast to previous studies, there was significantly less IL-10 (and more TNF-) secreted by restimulated T-bet–/– T cells compared with control T cells, findings which are unexpected in light of the diminished inflammatory effects of T-bet–/– cells in the heart. As expected, T-bet–/– cells secreted less IFN- than control cells, however, the quantity detected in the cultures of T-bet–/– cells was considerable. Similar results were obtained after anti-CD3-restimulation of control and T-bet–/– OT-I cells (data not shown). Real-time RT-PCR analyses of cytokine mRNAs in T-bet–/– and control OT-I cells (data not shown) correlated with the cytokine protein analyses.
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In vitro cytotoxicity assays showed significantly reduced killing of target cells by T-bet-deficient OT-I cells as compared with control OT-I cells (Fig. 4B). In addition, quantitative RT-PCR analyses show reduced expression in T-bet-deficient OT-I cells of genes involved in cytotoxic function, including granzyme B, perforin, and Fas ligand (Fig. 4C).
T-bet-deficient CD8+ effector T cells have an impaired migratory phenotype
We have shown that after adoptive transfer into cMy-mOVA mice, OT-I effector cells are detectable by 72 h in a peribronchial lymph node (17) that has the equivalent anatomic location of the cardiac lymph node identified in other species (28). In those studies, there were few OT-I cells still present in the cardiac lymph node 96 h after adoptive transfer, but at that time point, the T cells were detectable in large numbers in the myocardium. In the current study, we observed a similar pattern with few control OT-I cells detectable in the cardiac lymph node 96 h after transfer, but in contrast, abundant T-bet–/– OT-I cells were detectable in the lymph node at the same time point (Fig. 5A). This indicates that the lack of significant myocarditis in recipients of T-bet-deficient OT-I is not simply attributable to poor survival of these cells within their adoptive hosts. T-bet-deficient OT-I cells were also detectable in spleen and mesenteric lymph nodes (data not shown), albeit in lower percentages than in the cardiac lymph node. These findings suggest that T-bet–/– OT-I cells remain in the circulation longer than control T cells. Analysis of mRNAs for chemokine receptor genes demonstrated striking differences between T-bet–/– and control OT-I cells (Fig. 5B). CCR7 is a receptor which binds EB–/– ligand chemokine (CCL19) and secondary lymphoid tissue chemokine (CCL21), which are involved in lymph node retention of T cells (29), as well as the exit of T cells from peripheral tissues into afferent lymphatics (30). We found that T-bet–/– OT-I cells express significantly more CCR7 mRNA than do control OT-I cells. In contrast, compared with control OT-I cells, T-bet–/– OT-I cells express significantly less CXCR3, CCR2, and CCR5 mRNAs, which are receptors for several different chemokines that are commonly expressed in inflammatory sites. Consistent with the RNA data, we also found no CXCR3 protein detectable by flow cytometry on T-bet–/– cells, while CXCR3 protein was readily detectable on control cells (Fig. 5C).
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CXCR3-deficient CD8+ T cell effectors are impaired in their ability to migrate to the heart and cause myocarditis
On the basis of the data presented above, we hypothesized that small numbers of OT-I cells initially enter the heart in the absence of inflammatory chemokine expression and initiate myocardial damage and inflammation, which hastens recruitment of additional T cells and the development of severe disease. Because this inflammatory response clearly includes production of chemokines that bind to receptors expressed at much higher levels on wild-type than on T-bet–/– OT-I cells, we predicted that an isolated deficiency in one or more of those chemokine receptors would mimic the migratory defect observed in T-bet–/– OT-I cells. We chose to study the effects of CXCR3 deficiency because of the >28-fold reduction of this receptor on T-bet–/–OT-I cells vs control, as compared with more modest reductions in CCR5 and CCR2. CXCR3-deficient OT-I mice were generated by cross-breeding already established parental lines, and effector cells were generated as described earlier for wild-type OT-I cells. The CXCR3-deficient OT-I effector cells displayed a comparable activation phenotype and secreted similar amounts of inflammatory cytokines compared with control OT-I effector cells (data not shown), but showed impaired chemotactic responses to CXCR3 ligands, as expected (Fig. 6A). We found that CXCR3–/– OT-I cells were markedly less pathogenic than control OT-I cells in the cMy-mOVA mouse, as assessed by histopathology, serum TnI levels, and serum cytokine levels (Fig. 6, B–E). Further, the impaired pathogenicity of CXCR3-deficient OT-I cells is consistent with a defect in migration of these cells, rather than a defect in intrinsic effector function. Supportive of this view is the finding that at later time points of analysis and/or at higher doses of transferred T cells, pathogenicity differences between CXCR3-deficient and control OT-I effectors disappear (data not shown). This transient effect has also been reported previously in murine models of T cell recruitment to lung (31), and likely reflects the redundant function of other chemokine receptors in T cell recruitment.
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To clarify further whether the migratory defect of T-bet-deficient CD8+ T cells is due to a block in CXCR3 expression, we infected T-bet–/– OT-I cells with a human CXCR3-expressing retroviral vector. Human CXCR3-expressing mouse T cells respond to murine CXCR3 ligands (24), and the CXCR3 virus reconstitutes CXCR3 ligand-induced in vitro migration in CXCR3-deficient OT-I cells (data not shown). OT-I cells infected with the virus stained brightly for human CXCR3 (Fig. 7A). Histopathological appearance and scoring, as well as serum TnI determination indicated that CXCR3 virus-transduced T-bet–/– OT-I cells caused significantly more myocardial disease than control virus-infected T-bet–/– OT-I cells (Fig. 7, B–D). These data confirm the role CXCR3 plays in migration of CD8+ effector T cells into the myocardium, and establish that CXCR3 deficiency contributes greatly to the diminished pathogenicity of T-bet–/– OT-I cells. Nonetheless, although the mean histopathological score obtained after transfer of CXCR3-transduced T-bet–/– OT-I cells was 90% of that obtained with control-virus infected T-bet+/+ OT-I cells (3.6 vs 4.0, respectively), the serum TnI levels indicate that the degree of myocardial damage induced by the CXCR3-transduced OT-I cells was significantly less than damage induced by control OT-I cells (compare with Fig. 2B). Among serum cytokines measured, only IL-6 was significantly higher in recipients of CXCR3 virus-infected T-bet–/– OT-I cells compared with recipients of control virus-infected cells (Fig. 7E). Thus, forced CXCR3 expression resulted in significant reconstitution of migration of T-bet–/– OT-I cells into the heart, but only partial reconstitution of pathogenic phenotype of these cells. This was not because of nonspecific impairment of T cell effector function related to viral infection, because control OT-I cells infected with control virus induced comparably severe disease as uninfected control OT-I cells (data not shown).
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Another potential explanation for reduced pathogenicity of T-bet-deficient T cells is a relatively high susceptibility to regulatory mechanisms that normally control T cell-mediated immune responses. T-bet-deficient OT-I cells produce 100-fold more IL-2 than control OT-I cells (Fig. 2, and data not shown). One important regulatory function of IL-2 is to enhance development of regulatory T cells (Treg) of the CD4+CD25+FoxP3+ phenotype. Our original report of the cMy-mOVA model of myocarditis characterized the lymphocytic inflammatory infiltrate as predominately CD8+, with few endogenous CD4+ cells (17). Interestingly, immunohistochemical analysis of cMy-mOVA hearts from recipients of T-bet-deficient OT-I cells revealed increased ratios of CD4 to CD8 staining, compared with recipients of control T cells (data not shown). We therefore analyzed mRNA from hearts of cMy-mOVA recipients of OT-I cells to determine whether there were differences in expression of genes associated with Treg. Both FoxP3 and TGF- mRNA levels were significantly elevated in cMy-mOVA hearts 5 days posttransfer of T-bet-deficient OT-I cells, as compared with mRNA levels in recipients of control OT-I cells (Fig. 8A).
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secreted by the infiltrating T cells, and blockade of PD-L1 enhances the intensity of CD8+ T cell-mediated myocarditis (28). Interestingly, although PD-L1 expression is exceedingly low in the uninflamed heart, we detected significant PD-L1 expression in hearts of cMy-mOVA recipients of T-bet–/– OT-I cells by immunohistochemistry. Strikingly, quantification of PD-L1 by real-time RT-PCR revealed a 20-fold greater ratio of PD-L1 to IFN- expression in cMy-mOVA hearts 5 days posttransfer of T-bet-deficient vs control OT-I cells (Fig. 8B). Expression of PD-1 on the surface of OT-I cells was not significantly different between T-bet–/– and control groups before transfer (data not shown). Thus, the low-level inflammation induced by T-bet–/– OT-I effectors may actually result in protective responses by cardiac endothelia and myocytes. Additionally, T-bet-deficient OT-I cells expressed significantly more Fas and caspase 8 mRNA, and significantly less TIAP mRNA compared with control OT-I cells (Fig. 8C), consistent with enhanced susceptibility of T-bet-deficient T cells to mechanisms of apoptotic cell death.
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The differentiation of naive T cells induced by primary exposure to Ag involves not only the expression of molecules involved in effector function, but also the expression of adhesion molecules and chemokine receptors that are required for efficient migration of effector T cells into inflammatory sites. The current study is the first to demonstrate that T-bet-dependent CXCR3 expression is required for pathogenicity of CD8+ T cells. Specifically, we show that T-bet regulates the migratory phenotype of CD8+ effector T cells, and importantly, that this aspect of T-bet function profoundly influences the capability of tissue Ag-specific CD8+ T cells to cause disease in the heart. In particular, we found a down-regulation of CXCR3, CCR5, and CCR2, and up-regulation of CCR7 in T-bet-deficient CD8+ T cells. The relatively low level of expression of CCR5 is also a characteristic of CD8+ effector T cells differentiated in the absence of exogenous IL-12 (17). Both the T-bet null OT-I cells and the IL-12-deprived OT-I cells are far less pathogenic than wild-type, IL-12-treated OT-I cells in the cMy-mOVA myocarditis model, and we have demonstrated that the ligands for both CXCR3 and CCR5 are expressed in cMy-mOVA hearts during the course of OT-I-mediated myocarditis. Therefore, two independent methods of altering differentiation of effector CD8+ T cells result in reduced expression of inflammatory chemokine receptors, although IL-12 deprivation appears to have a more selective effect. We found approximately equal levels of IL-12R 2 chain mRNA in both wild-type and T-bet-deficient OT-I cells (data not shown), and therefore the effect of T-bet deficiency on the migratory phenotype of the cells is not simply due to a lack of IL-12R expression.
The relatively high level of CCR7 expression seen in T-bet-deficient CD8+ T cells is also a characteristic of CD8+ effector T cells differentiated in the absence of exogenous IL-12 (17). This persistent expression of CCR7 on these less pathogenic cells is consistent with their prolonged presence in the cardiac lymph node (Fig. 6), because CCR7 is known to direct CD8+ T cell migration through high endothelial venules into lymph node parenchyma (34). The failure to see wild-type OT-I cells in the cardiac lymph node at 96 h may reflect the loss of expression of CCR7 on those cells, or their sequestration in the heart.
The cMy-mOVA mouse heart is uninflamed at the time of adoptive transfer of OT-I cells, with little or no detectable chemokine expression. Nonetheless, our data indicate that CXCR3 expression is required for significant infiltration of CD8+ T cells into the heart. The most likely explanation for this is that the CD8+ effector T cells have a limited capacity to enter uninflamed myocardium, albeit in numbers that are insufficient to cause detectable damage. There is evidence of constitutive low level migration of effector T cells into normal tissues (11). It is possible that endothelial cross-presentation of OVA produced by myocytes may enhance OT-I-selective recruitment. Once the OT-I effector cells encounter OVA-expressing myocardial cells, they will become reactivated and cause myocyte damage. These early events would initiate a self-amplifying inflammatory cascade that includes the production of IFN--inducible chemokines that serve to enhance recruitment of CXCR3-expressing T cells. The mobilization of Ag (OVA) from damaged myocardium, which will drain into the cardiac lymph node, is likely to further amplify the OT-I response and the inflammatory process. Our analysis of chemokine gene expression in the myocardium, and the kinetics of lymph node appearance of OT-I cells are consistent with this model. Most significantly, the diminished pathogenicity of CXCR3-deficient OT-I cells, and the enhanced myocardial recruitment of T-bet-deficient OT-I cells after CXCR3-viral transduction, support the hypothesis that chemokine-dependent T cell recruitment becomes critical for sustained disease.
Another possible effect of T-bet deficiency that may contribute to the diminished migratory capabilities of OT-I effector cells is the impaired expression of functional selectin ligands. We have previously shown that fucosyltransferase-dependent selectin ligand synthesis contributes to the ability of OT-I effectors to enter hearts of cMy-mOVA mice (23). Although T-bet deficiency results in diminished CD4+ Th1 cell binding to P-selectin (15), it is not clear whether T-bet-deficient CD8+ T cells also have diminished ability to bind P-selectin. Nonetheless, our data indicate that the CD8+ T cells migratory defect associated with T-bet deficiency is largely corrected by ectopic CXCR3 expression.
Our ability to measure serum troponin levels (using a sensitive clinical assay not previously applied to mouse studies) has allowed us to monitor myocyte damage independent of T cell recruitment into the heart. After adoptive transfer of competent (wild-type) effector OT-I cells, there is a good correlation between the serum TnI levels and histopathological scores (see Fig. 1), establishing the validity of the TnI assay to assess disease. In contrast, the TnI levels attained in mice receiving CXCR3 virus-infected T-bet-deficient OT-I cells were only a small fraction of those attained after wild-type OT-I transfer, even though the histopathological scores in the two groups were similar. The discrepancy between histopathological score and serum TnI levels after CXCR3 reconstitution indicates that, even after enhancing migratory ability, T-bet-deficient OT-I cells were not fully capable of injuring myocardial cells. Thus, the data demonstrate the distinct contributions T-bet plays in regulating migratory ability and cytotoxicity, both required for the pathogenic phenotype of CD8+ effectors. Further studies will be required to evaluate how T-bet influences naive OT-I differentiation and migration in cMy-mOVA mice following transfer and immunization.
An additional explanation for reduced pathogenicity of T-bet-deficient cells is supported by data indicating these cells have higher susceptibility to both extrinsic and intrinsic regulatory mechanisms. It is plausible that activated T-bet-deficient OT-I cells, which express high levels of CCR7 and low levels of CXCR3 (Fig. 5B), remain largely in the lymphatic circulation, accumulating in the peribronchial cardiac lymph node while secreting elevated levels of IL-2 and low levels of IFN- (Fig. 4A), which may significantly alter the local Treg milieu. Those cells that do migrate to the heart may thus encounter increased levels of Treg and PD-L1 relative to the level of inflammation present (Fig. 8, A and B), leading to dramatically reduced myocyte damage by cells which may already express higher levels of proapoptotic proteins at baseline (Fig. 8C).
In summary, our results establish the importance of T-bet in the differentiation of several phenotypic characteristics which enhance the ability of CD8+ T cells to enter the heart and cause lethal myocardial injury. Our findings specifically link, for the first time, T-bet with the differentiation of cardiopathogenic T cells, and suggest potential targets for therapeutic intervention of CTL-mediated cardiac disease, including CXCR3-dependent migratory mechanisms.
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Disclosures |
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1 This work was supported by the following National Institutes of Health Grants AI059610 and HL072056 (to A.H.L.); CA48126 and AI56296 (to L.H.G.); and DK074449 and CA69212 (to A.D.L. and R.C.). Additional support came from The Roche Organ Transplantation Research Foundation (to A.D.L.), Howard Hughes Medical Institute Research Training Fellowship, American Medical Association Seed Grant, and American Academy of Allergy, Asthma, and Immunology (to V.R.T.). 
2 Address correspondence and reprint requests to Dr. Andrew H. Lichtman, Department of Pathology, Brigham and Women’s Hospital, NRB-7, Room 752N, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: alichtman{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: EGFP, enhanced GFP; TnI, troponin I; PD-L1, programmed death-ligand 1; TIAP, thymus inhibitor of apoptosis; Mig, monokine induced by IFN-; I-TAC, IFN-inducible T cells chemoattractant; IP-10, IFN-inducible protein 10; Treg, regulatory T cell.
Received for publication June 7, 2006. Accepted for publication August 7, 2006.
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+ cells and dominance of granzyme A+, CD8+ lymphocytes. Am. J. Trop. Med. Hyg. 48: 637-644. -1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands. J. Immunol. 167: 4476-4484. on the extent and phenotype of diet-induced atherosclerosis in the LDLR-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 23: 454-460. production in CD4 and CD8 T cells. Science 295: 338-342. This article has been cited by other articles:
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J. Barbi, H. E. Cummings, B. Lu, S. Oghumu, T. Ruckle, C. Rommel, W. Lafuse, C. C. Whitacre, and A. R. Satoskar PI3Kgamma (PI3K{gamma}) is essential for efficient induction of CXCR3 on activated T cells Blood, October 15, 2008; 112(8): 3048 - 3051. [Abstract] [Full Text] [PDF]
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J. Barbi, F. Brombacher, and A. R. Satoskar T Cells from Leishmania major-Susceptible BALB/c Mice Have a Defect in Efficiently Up-Regulating CXCR3 upon Activation J. Immunol., October 1, 2008; 181(7): 4613 - 4620. [Abstract] [Full Text] [PDF]
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J. A. Lucas, J. Menke, W. A. Rabacal, F. J. Schoen, A. H. Sharpe, and V. R. Kelley Programmed Death Ligand 1 Regulates a Critical Checkpoint for Autoimmune Myocarditis and Pneumonitis in MRL Mice J. Immunol., August 15, 2008; 181(4): 2513 - 2521. [Abstract] [Full Text] [PDF]
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