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TGF-1 Regulates Antigen-Specific CD4⁺ T Cell Responses in the Periphery¹

Department of Pathology and Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756

	Abstract

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T cell expansion typically is due to cognate interactions with specific Ag, although T cells can be experimentally activated through bystander mechanisms not involving specific Ag. TGF-1 knockout mice exhibit a striking expansion of CD4⁺ T cells in the liver by 11 days of age, accompanied by CD4⁺ T cell-dependent necroinflammatory liver disease. To examine whether hepatic CD4⁺ T cell expansion in TGF-1^–/– mice is due to cognate TCR-peptide interactions, we used spectratype analysis to examine the diversity in TCR V repertoires in peripheral CD4⁺ T cells. We reasoned that Ag-nonspecific T cell responses would yield spectratype profiles similar to those derived from control polyclonal T cell populations, whereas Ag-specific T cell responses would yield perturbed spectratype profiles. Spleen and liver CD4⁺ T cells from 11-day-old TGF-1^–/– mice characteristically exhibited highly perturbed nonpolyclonal distributions of TCR V CDR3 lengths, indicative of Ag-driven T cell responses. We quantitatively assessed spectratype perturbation to derive a spectratype complexity score. Spectratype complexity scores were considerably higher for TGF-1^–/– CD4⁺ T cells than for TGF-1^+/– CD4⁺ T cells. TCR repertoire perturbations were apparent as early as postnatal day 3 and preceded both hepatic T cell expansion and liver damage. By contrast, TGF-1^–/– CD4⁺ single-positive thymocytes from 11-day-old mice exhibited normal unbiased spectratype profiles. These results indicate that CD4⁺ T cells in TGF-1^–/– mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-1 in the peripheral regulation of Ag-specific CD4⁺ T cell responses.

	Introduction

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Transforming growth factor-1 is an important immunomodulatory cytokine that regulates the differentiation, proliferation, and apoptosis of CD4⁺ T cells, but the mechanisms by which TGF-1 regulates T cell homeostasis remain incompletely understood (1). Mice homozygous for a targeted deletion in the gene encoding TGF-1 (TGF-1^–/– mice) develop a striking peripheral organ T cell lymphocytosis within a few weeks of birth, as well as inflammatory pathologic changes in several organs, primarily the liver, lungs, and heart (2, 3). On the BALB/c background, TGF-1^–/– mice develop a marked necroinflammatory liver disease, with a striking hepatic T cell lymphocytosis, accompanied by lymphocyte-mediated organ destruction within 2 wk of birth (4). The pathogenic role of CD4⁺ cells in the phenotype of TGF-1^–/– mice is well established, because depletion of the CD4⁺ T cell subset prevents the development of necroinflammatory liver disease (5). Moreover, MHC class II^–/–TGF-1^–/– double-deficient mice, in which mature CD4⁺ T cells fail to develop, do not develop inflammatory disease (6).

The factors involved in the initiation of the inflammatory response in TGF-1^–/– mice are currently unknown. TGF-1 has an enormously broad spectrum of effects in the immune system, with important influences on multiple cell types and multiple cell processes, including pathways that regulate cell activation, proliferation, differentiation, signaling, adhesion, migration, and apoptosis (7). Therefore, it is not obvious a priori that the CD4⁺ T cell response in TGF-1^–/– mice must be due to the interaction of T cells with specific Ags. Indeed, given the pleiotropic effects of TGF-1, one reasonable model is that TGF-1 plays a key role in dampening "background" homeostatic interactions of CD4⁺ T cells with their environment (including interactions with cytokines, adhesive molecules, or MHC molecules). Perhaps, in the absence of this key regulatory cytokine, these background interactions of T cells are sufficiently strong to cause T cell activation and expansion.

Some studies examining the pathogenesis of disease in TGF-1^–/– mice implicate a role for peripheral autoantigens in driving T cell expansion and the development of autoimmune disease. The majority of CD4⁺ T cells in the liver and spleen from TGF-1^–/– mice express low levels of CD62 ligand (CD62L)³ and high levels of CD44 and VLA-4, a surface expression profile consistent with TCR activation (4), although LPS alone can also induce similar T cell surface expression profiles (8). Evidence in support of the specific Ag model has been published recently; experimentally restricting the TCR repertoire available to CD4⁺ T cells in TGF-1^–/– mice, through breeding of the TGF-1^–/– mouse with the OVA-specific DO11.10 TCR-transgenic mouse, prevented the development of T cell activation and tissue pathology (9, 10). Further implicating a pathologic role for self-Ags, TGF-1^–/– mice raised in germfree conditions nevertheless develop end-organ inflammatory pathology, indicating that the presence of intestinal microflora is not necessary for disease (11).

Under certain circumstances, T cells can be activated through bystander mechanisms that do not involve specific Ag signaling through the TCR (12). Ag-independent activation of both naive and memory resting T cells can be elicited in vitro using a combination of IL-2, TNF-, and IL-6 (13). In vivo, activation of TLR molecules causes widespread transient activation of both naive and memory T cells (8). T cells can also be activated through the ligation of CD2 (14, 15), a pathway that bypasses the requirement for specific Ag recognition. Furthermore, during acute inflammatory responses, T cell-mediated tissue damage may develop through mechanisms that do not necessarily involve direct interaction of T cells with tissue Ag. This is particularly relevant for the liver, where experimental model systems establish that hepatocellular damage can be induced after T cell activation and migration to the liver, even when cognate Ag is not expressed in the liver (16, 17, 18). Such mechanisms may involve the elaboration of inflammatory cytokines, such as IFN- and TNF-, and the production of soluble Fas ligand. Both IFN- and TNF- are toxic to hepatocytes (19, 20, 21); Fas is constitutively expressed on hepatocytes and its engagement rapidly causes hepatocyte apoptosis (22, 23). Livers from TGF-1^–/– mice exhibit high levels of expression of mRNAs encoding IFN-, TNF-, and Fas ligand, suggesting that the elaboration of these molecules may participate in the disease process, and indeed TGF-1^–/–/IFN-^–/– double knockout mice are protected from the development of necroinflammatory liver disease (4).

In this study, we used the PCR-based spectratype technique, which assesses the distribution of CDR3 lengths in V genes in a T cell population, to examine the diversity of the TCR repertoire in CD4⁺ T cells from TGF-1^–/– mice. We reasoned that Ag-nonspecific T cell responses would yield polyclonal spectratype profiles similar to profiles derived from T cells from control mice, whereas Ag-specific T cell responses would yield oligoclonal spectratype profiles that deviate from the profiles from a normal polyclonal population. Just such a dichotomy has been reported in spectratype analyses of two models of spontaneous T cell expansion and pathology: T cells spontaneously expand in the periphery of both CTLA4^–/– mice and Jak3^–/– mice. CTLA4^–/– mice yield normal spectratype profiles, reflecting polyclonal, uniform expansion of all CD4⁺ T cells, regardless of Ag specificity, whereas Jak3^–/– mice exhibit highly abnormal spectratype profiles, reflecting oligoclonal, preferential expansion of some T cells but not others (24).

In the current study, we found that liver CD4⁺ T cells from TGF-1^–/– mice characteristically exhibit strongly skewed nonpolyclonal distributions of CDR3 lengths, indicative of Ag-driven immune responses. We applied a kinetic, quantitative analysis of spectratype profiles to show that the development of a skewed TCR repertoire in liver CD4⁺ T cells precedes both T cell expansion and the onset of detectable end-organ damage, with TCR repertoire deviations apparent as early as postnatal day 3. Moreover, whereas TGF-1^–/– peripheral CD4⁺ T cells exhibited strikingly abnormal CDR3 length distributions, TGF-1^–/– CD4⁺ single-positive (CD4SP) thymocytes exhibited unbiased, polyclonal CDR3 length distributions. These data indicate that CD4⁺ T cells in TGF-1^–/– mice are activated by self-Ags present in the periphery, and that a key immunoregulatory role for TGF-1 is to prevent self-Ags from activating peripheral CD4⁺ T cells and triggering the development of inflammatory tissue pathology.

	Materials and Methods

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Mice

Mice were bred at the Dartmouth Medical School American Association for Accreditation of Laboratory Animal Care-accredited animal care facility and were treated humanely according to National Institutes of Health guidelines. The derivation of BALB/c TGF-1^+/– mice is published (4). Litters were screened by the PCR of DNA from tail snips for TGFB1 genotypes as described (25).

Isolation of lymphocytes and thymocytes

TGF-1^–/– mice and littermate control TGF-1^+/– mice were euthanized by overexposure to CO₂. Mice were cardiac perfused with PBS, and mononuclear cells (MNC) from the liver, lung, spleen, and thymus were isolated. Organs were dissected out, weighed, and homogenized in ice-cold 2% FCS/RPMI 1640 using a Powergen Tissue Homogenizer (Fisher Scientific) set at its lowest speed. Homogenized livers and lungs were washed with serum-free RPMI 1640 and resuspended in 5 ml of 100U/ml collagenase IV (Sigma-Aldrich) and 10 U/ml DNase I (Roche) in serum-free RPMI 1640. The organ/digestion solution mixture was incubated at 37°C for 45 min with inverting every 2 min. After washing with cold 2% FCS/RPMI 1640, the cells from each organ were resuspended in room temperature 2% FCS/RPMI 1640 and collected over Ficoll (Sigma-Aldrich). MNC were counted and either stained for flow cytometric analysis or used for positive selection of CD4⁺ cells (for the spleen, lung, and liver) or CD8⁺ negative selection (for thymus) to obtain CD4SP cells using the Miltenyi Biotec system.

Serum transaminase analysis

Directly following euthanasia, blood was collected into heparin-coated plasma separator tubes (BD Biosciences) from the right atrium of the heart before perfusion. Plasma was diluted 5-fold and alanine aminotransferase (ALT) levels were determined on a Roche-Hitachi 917 Automatic Analyzer using a spectrophotometric assay.

V usage analysis by flow cytometry

All Abs used for flow cytometric analysis were purchased from BD Pharmingen. Following their collection over Ficoll, MNC were washed with 2% FCS in PBS (FACS buffer) and stained with fluorochrome-labeled Abs that recognize CD3, CD4, and the CD8 chain (clones 17A2, RM4-5, and 53-6.7, respectively), as well as 1 of a panel of 15 FITC-labeled mAbs (BD Pharmingen Mouse V Screening Panel) that recognize surface-expressed TCR V chains according to the manufacturer’s instructions. TCR V chains recognized by this panel include V 2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 8.3, 9, 10, 11, 12, 13, 14, and 17. Cells were washed with FACS buffer and fixed in 1% methanol-free formalin in FACS buffer. Acquisition of stained cells was done using a FACSCalibur cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences). For all surface markers, positive staining was established using appropriate isotype controls.

RNA purification and cDNA synthesis

Total RNA was isolated from the indicated cell populations using the RNeasy method (Qiagen). cDNA was synthesized using the Omniscript reverse transcription PCR kit (Qiagen) with oligo(dT) primers (Roche).

Spectratype analysis

Spectratype analysis was modified from Ref. 26 . In brief, CDR3 regions of V-specific cDNAs were first amplified by PCR in a 10-µl reaction comprising the following: 10 mM Tris-Cl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 200 µM dNTPs, 0.25 U Taq polymerase, 1 µM C1/C2 reverse primer, 1 µM V-specific forward primer, and a minimum of 200 pg cDNA. V-containing amplicons were fluorescently labeled by runoff PCR; 2.5 µl of the initial amplification reaction was labeled in a final volume of 10 µl in the following reaction conditions: 10 mM Tris-Cl (pH 8.3), 1.5 mM MgCl₂, 50 mM KCl, 200 µM dNTPs, 0.25 U Taq polymerase, 2and 00 nM C1/C2 FAM-labeled nested reverse primer. All primer sequences are as published previously (26), except for the following: V8.1, 5'-GGCTGATTCATTACTCATATGTC-3'; V8.2, 5'-TCATATGGTGCTGGCAGCACTG-3'; V13, 5'-AGGCCTAAAGGAACTAACTCCACT-3'; and V17, 5'-CTAAGTGTTCCTCGAACTCAC-3'. To each reaction tube, 1.0 µl of ROX 400HD size standard (Applied Biosystems) and 11 µl of HiDi Formamide (Applied Biosystems) were added. Following denaturation, products were detected and sizes were determined using an Applied Biosystems 3100 sequencer and GeneScan software (Applied Biosystems). Spectratype profiles were successfully obtained for all Vs expressed from the BALB/c genome; no profiles were obtained for V5.3 and V9, which are not expressed on BALB/c. Quantification of spectratype profiles was determined by the spectratype complexity (SC) score, computed essentially as described in Ref. 27 , and described in more detail in Results.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TCR V usage in liver TGF-1^–/– CD4⁺ T cells

BALB/c background TGF-1^–/– mice develop a dramatic hepatic CD4⁺ T cell lymphocytosis with accompanying severe liver damage by 10–11 days of age (4). The accumulation of CD4⁺ T cells in liver may be due either to an expansion of a population of CD4⁺ T cells expressing a full repertoire of TCR V-chains, or to an expansion of a CD4⁺ T cell population expressing only one or a few TCR V chains. To distinguish between these possibilities, we used flow cytometry to examine TCR V expression on hepatic CD4⁺ T cells from 11-day-old mice using a panel of mAbs that assesses 15 of the 20 possible murine TCR V chains. TGF-1^–/– CD4⁺ T cells accumulated in liver, regardless of specific TCR V-chain expression, although there was variability in the degree of expansion among specific TCR V-expressing CD4⁺ T cell subsets (Fig. 1). Thus, T cell expansion in the liver occurs broadly with respect to TCR V chain usage.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Accumulation of CD4+ T cells in TGF-1–/– liver occurs broadly with respect to TCR V chain usage. Intrahepatic MNC were isolated and stained for CD3, CD4, CD8, and each TCR V chain using a panel that assesses 15 of the 20 possible TCR V chains. The percentage of TCR V expression was determined after gating for CD3+CD4+CD8– cells, and the number of V-expressing T cells was determined by multiplying this number by the calculated total number of CD4+ T cells per liver. TGF-1–/– mice, n = 4. TGF-1+/– littermate control mice, n = 6. Bars, Mean + 1 SD.

TCR V transcripts from TGF-1^–/– CD4⁺ T cells exhibit skewed CDR3 size distributions

The broad expansion of CD4⁺ T cells from all analyzed TCR V-expressing subsets suggests that the observed end-organ lymphocytosis is a polyclonal phenomenon. To examine T cell clonality with greater sensitivity, we used spectratype analysis to assess CDR3 lengths in TCR V transcripts from CD4⁺ T cells (see Materials and Methods). For a given TCR V gene, CDR3 size varies between T cells, owing to nucleotide additions catalyzed by the enzyme TdT at the TCR somatic gene rearrangement during T cell ontogeny. TdT activity results in the addition of 12–52 nt, corresponding to an addition of 4–13 aa in the translated TCR- protein. For a given specific TCR V-containing transcript, spectratype analysis of a normal naive T cell population yields a Gaussian distribution of CDR3 sizes that differ by 3 nt. By contrast, spectratype analysis of specific TCR V-containing transcripts from a nonpolyclonal T cell population yields a non-Gaussian distribution characterized by one or more spikes in the profile. As an example, Fig. 2A shows spectratype profiles for V8.2 as applied to a polyclonal sample (MNC from a wild-type BALB/c mouse) and to a monoclonal sample (MNC from a DO11.10 TCR-transgenic mouse), respectively.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. TCR V spectratypes in TGF-1–/– CD4+ T cell populations are perturbed. A, Left, Example of a typical spectratype profile for MNC from a BALB/c wild-type mouse, using primers that amplify the CDR3 region of V8.2-containing transcripts. A Gaussian distribution of peaks is observed. Peaks differ by 3 nt (1 aa in the translated protein), with a maximum at 24 nt (i.e., 8 aa). Right, V8.2 spectratype profile for MNC from a DO11.10 mouse showing a single prominent peak, as expected for this clonal T cell population. B, Representative spectratype profiles applied to spleen CD4+ T cells or liver CD4+ T cells from two 11-day-old TGF-1+/– mice and two 11-day-old TGF-1–/– mice. Results were obtained using primers that amplify CDR3 regions from the TCR V genes indicated.

Quantitative assessment of TCR V CDR3 size distributions shows large differences between TGF-1^–/– CD4⁺ T cells and TGF-1^+/– CD4⁺ T cells

To minimize the introduction of bias to our analysis, we identified a methodology to analyze spectratype profiles that permits the quantification for a given spectratype profile of the deviation from normal. The SC score (determined as in Ref. 27) calculates the aggregate difference between CDR3 lengths for a given V transcript profile, and those from a collection of profiles from a reference population. In brief, the area under the curve for each peak is determined and represented as a frequency of the total (Fig. 3A). The application of this analysis to CD4⁺ T cell samples from four TGF-1^+/– mice (the reference population) generates a histogram that represents the percentage use of each CDR3 length for a given V gene segment. The profile for an individual test sample is then compared with the generated distribution of the reference population (Fig. 3B), and the differences in individual peak frequencies are summed to yield the SC score (Fig. 3C). Higher SC scores represent greater deviations from a reference population. As a benchmark, SC score analysis for TCR V8.2 applied to a DO11.10 sample compared with a reference population sample yielded an SC score of 144.1 (arbitrary units; data not shown), which approximates a maximum possible SC score for a completely monoclonal T cell population.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Method of determination of SC score. A, For a given TCR V spectratype profile, the area under the curve for each peak was determined and calculated as a frequency (in percentages) of the total area for all peaks. To generate a reference population histogram (shown here for TCR V12), this analysis is performed for samples prepared from four individual TGF-1+/– mice. Bars, Mean + 1 SD. For test samples, frequencies for each peak were determined the same way. B and C, The distribution of frequencies for a given test sample was then compared with the distribution of mean frequencies derived from the reference population. This comparison is depicted visually (B) and is the basis for the formula deriving the SC score. C, The SC score represents the summation of the absolute value of differences in frequencies of each peak between the sample spectratype profile and the reference spectratype profile.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. SC score analyses of TGF-1–/– CD4+ T cells. SC scores were determined for eight different TCR V spectratypes (V4, V6, V7, V8.1, V8.2, V10, V11, and V12) from TGF-1+/– spleen CD4+ T cells (A), TGF-1–/– spleen CD4+ T cells (B), TGF-1+/– liver CD4+ T cells (C), and TGF-1–/– liver CD4+ T cells (D); n = 3–4 mice per analysis. Horizontal lines, mean SC scores. Shown above B and D are p values calculated from statistical analyses of SC scores for individual gene segments from TGF-1–/– samples and their TGF-1+/– counterparts in A or C, respectively. Statistical analyses used Student’s t test.

The development of perturbations in the TGF-1^–/– CD4⁺ TCR V repertoire precedes CD4⁺ T cell accumulation in liver and end-organ damage

We asked whether the development of a skewed TCR repertoire precedes T cell expansion and end-organ damage. To address this, we conducted a kinetic analysis of T cell CDR3 length usage using the quantitative SC score as a metric. For this analysis, we averaged the SC scores from all V gene segments profiled for each T cell sample (n = 7–8 V gene segments) to develop a composite SC score. Composite SC scores were determined from spleen and liver CD4⁺ T cells from TGF-1^–/– mice and littermate control TGF-1^+/– mice at 3–4 days of age, 7 days of age, and 10–11 days of age. Fig. 5A shows representative spectratype profiles obtained for a very young (3 day) TGF-1^–/– mouse and its TGF-1^+/– littermate; profiles from both mice are similar, exhibiting largely Gaussian CDR3 distributions for all V gene segments analyzed (Fig. 5A). For TGF-1^–/– mice at 3–4 days of age (n = 3), calculated composite SC scores determined for splenic CD4⁺ T cells were similar (p = 0.3) to calculated composite SC scores determined for splenic CD4⁺ T cells obtained from littermate control TGF-1^+/– mice (n = 7). For TGF-1^–/– mice at 7 days of age (n = 3), calculated composite SC scores determined for splenic CD4⁺ T cells were higher than at 3 days of age, and were significantly different (p = 0.01) from composite SC scores determined for splenic CD4⁺ T cells from littermate TGF-1^+/– mice (n = 4). For TGF-1^–/– mice at 10–11 days of age (n = 6), the calculated composite SC scores determined for splenic CD4⁺ T cells increased further, and were significantly different (p < 0.0001) from composite SC scores obtained for splenic CD4⁺ T cells from littermate TGF-1^+/– mice (n = 6; Fig. 5B). Thus, in TGF-1^–/– mice, TCR repertoires for splenic CD4⁺ T cells appear normal at 3–4 days of age, but become measurably perturbed by 7 days of age.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. TGF-1–/– liver CD4+ T cell repertoires are abnormal as early as postnatal day 3. A and C, CD4+ cells were isolated from the spleen (A) and liver (C) of a 3-day-old TGF-1–/– mouse and a littermate control TGF-1+/– mouse, and spectratype profiles were obtained as shown. B and D, Composite SC scores (from seven to eight individual TCR Vs) for spleen (B) and liver (D) CD4+ T cells were calculated and graphed as a function of age (3–4 days, 7 days, and 10–11 days of age); n = 3–7 mice per data point. The p values for each age (TGF-1–/– vs TGF-1+/–) are shown at the bottom of each graph. For each day range analyzed, the reference spectratype profile was generated from analyses of age- and organ-matched CD4+ T cell populations isolated from three to four additional individual TGF-1+/– mice. Symbols, Mean ± 1 SD.

Next, we examined CD4⁺ T cell numbers and assessed hepatocellular damage. The numbers of liver effector/memory (i.e., CD62L^low) CD4⁺ T cells in TGF-1^–/– mice and littermate control TGF-1^+/– mice were similar at days 3 and 4 and day 7, but at days 10 and 11, the numbers of liver CD62L^lowCD4⁺ T cells were elevated >10-fold in TGF-1^–/– mice compared to TGF-1^+/– mice (p = 0.008; Fig. 6A). Seventy-seven to 99% of CD4⁺ T cells from TGF-1^–/– livers were CD62L^low, whereas 54–86% of CD4⁺ T cells from TGF-1^+/– livers were CD62L^low (data not shown). Accumulation of CD4⁺ T cells was not unique to the liver; CD62L^lowCD4⁺ T cells accumulated in the TGF-1^–/– lung as well and with kinetics similar to the liver (Fig. 6B). Liver damage was determined by measuring plasma levels of the hepatocyte enzyme ALT. ALT levels were similar in TGF-1^–/– mice and TGF-1^+/– mice at or before postnatal day 7. At 10 and 11 days of age, ALT levels were elevated in TGF-1^–/– mice compared to TGF-1^+/– mice (p < 0.0001; Fig. 6C), a kinetic pattern consistent with our previously published studies (5). Thus, in TGF-1^–/– mice, the development of perturbations in the CD4⁺ T cell repertoire is detectable in the liver at 3–4 days of age, in the spleen at 7 days of age, and precedes both CD4⁺ T cell accumulation in the liver and lungs and the onset of detectable liver damage.

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Liver CD4+ T cell accumulation and liver disease in TGF-1–/– mice begins between postnatal days 7 and 10. TGF-1–/– mice and littermate control TGF-1+/– mice were sacrificed at the ages indicated. The numbers of CD62Llow CD4+ T cells in the liver (A) and lung (B) at each age are shown. CD4+ T cell number data were acquired from two to six mice per data point. C, Plasma ALT levels from mice sacrificed at each age are shown. ALT data were acquired from 3 to10 mice per data point for TGF-1–/– mice and from 5 to 18 mice per data point for TGF-1+/– mice. Symbols, Mean ± 1 SD. The p values for each age (TGF-1–/– vs TGF-1+/–) are shown at the bottom of each graph.

TGF-1^–/– CD4⁺ TCR V repertoire perturbations develop in the periphery, not in the thymus

This kinetic analysis suggests that the acquisition of a perturbed TCR repertoire is a largely peripheral phenomenon and allows the prediction that the thymic development of the CD4⁺ TCR repertoire is normal in TGF-1^–/– mice. We tested this hypothesis directly by simultaneously examining at the peak of necroinflammatory liver disease (11 days of age) peripheral CD4⁺ T cells and thymic CD4SP CD4⁺ T cells from TGF-1^–/– mice. Thymi from TGF-1^–/– mice were hypocellular (Fig. 7A) compared with littermate control TGF-1^+/– thymi, but the densities of CD4^–CD8^– double-negative, CD4⁺CD8⁺ double-positive, CD8⁺ single-positive, and CD4SP subsets were similar (Fig. 7B). The distribution of frequencies of TCR V chain usage among CD4SP thymocytes was not affected by the absence of TGF-1 (Fig. 7C). Spectratype analyses of CD4SP thymocytes yielded Gaussian profiles, regardless of whether cells were obtained from TGF-1^+/– mice or from TGF-1^–/– mice (Fig. 7D). By contrast, spectratype analyses of splenic CD4⁺ T cells isolated from the same mice yielded abnormal non-Gaussian profiles for TGF-1^–/– mice, but not for TGF-1^+/– mice. Thus, the development of perturbations in the CD4⁺ TCR V repertoire in TGF-1^–/– mice takes place in the periphery, not in the thymus.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. TCR repertoires are normal in TGF-1–/– CD4SP thymocytes. A, Thymi were isolated from two mice of each genotype at postnatal day 11 and weighed. B, Cellular densities of thymocyte subsets in 11-day-old mice were calculated from flow cytometric profiles. Two individual mice from each genotype were assessed. DN: CD4–CD8– double-negative cells; DP: CD4+CD8+ double-positive cells; CD8SP: CD8+ single-positive cells. C, TCR V usage (expressed as percentages) was determined in CD4SP thymocytes from 11-day-old TGF-1+/– mice (n = 3) and TGF-1–/– mice (n = 3). Bars, Mean + SD. D, Spectratype profiles were obtained for CD4SP thymocytes (top) or splenic CD4+ T cells (bottom) simultaneously isolated from littermate 11-day-old TGF-1+/– mice and TGF-1–/– mice. Results are representative of three mice from each genotype.

	Discussion

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TCR repertoire perturbations develop early in TGF-1^–/– mice

This work describes important events in the pathogenesis of autoimmune disease in TGF-1^–/– mice. We used the spectratype technique to analyze in CD4⁺ T cells the distribution of sizes of CDR3 hypervariable regions within TCR V genes. We applied a quantitative analysis of profiles obtained using spectratype to demonstrate that perturbations in the TGF-1^–/– CD4⁺ TCR repertoire become detectable in liver as early as postnatal day 3. Thereafter, liver CD4⁺ T cells exhibit a progressive increase in TCR repertoire deviations. Similar TCR repertoire abnormalities become detectable in splenic CD4⁺ T cells by day 7. Hepatic CD4⁺ T cell lymphocytosis becomes manifest by days 10 and 11, accompanied by widespread hepatocellular damage. At this age, the peripheral CD4⁺ T cell repertoire is severely perturbed. Importantly, the TCR repertoire in CD4SP thymocytes examined simultaneously is normal. Thus, the critical role of TGF-1 in modulating T cell homeostasis and preventing autoimmune liver disease is to regulate the peripheral T cell response rather than T cell ontogeny.

The very early deviation of the TCR repertoire in liver CD4⁺ T cells (at day 3) constitutes the earliest known abnormality of the adaptive immune system in TGF-1^–/– mice. Previously, Geiser et al. (29) showed that overexpression of class II mRNA in the liver and other peripheral organs in TGF-1^–/– mice is detectable at day 6, but not at day 3. Thus, TCR deviation precedes class II mRNA overexpression. Microarray gene expression analyses from our laboratory show that class II mRNA expression in day 11 TGF-1^–/– livers requires an intact IFN- gene (R. T. Robinson and J. D. Gorham, unpublished data). We also have evidence that the principal source of IFN- in TGF-1^–/– livers is the CD4⁺ T cell population, with little contribution from either NK cell or CD8⁺ T cell populations (R. T. Robinson and J. D. Gorham, unpublished data). Thus, we propose that activation of CD4⁺ T cells, with production of the effector cytokine IFN-, is antecedent to, and probably required for, class II up-regulation.

TGF-1 regulates Ag-specific peripheral CD4⁺ T cell responses

Expansion of T cells in the periphery can be either Ag driven or independent of Ag. Ag-specific stimulation requires TCR ligation by the peptide in the context of MHC class II molecules. Mechanisms mediating nonspecific stimulation of T cells include cytokine-driven responses and signals delivered through adhesive interactions (e.g., LFA-3/CD2). Illustrating the dichotomy between these two types of T cell responses is a published comparative analysis of TCR repertoire diversity in T cells from two different mouse models, both characterized by peripheral T cell activation and expansion. The tyrosine kinase Jak3 transduces signals mediated by the common -chain, a common component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mice homozygous for a targeted deletion in the gene encoding Jak3 develop CD4⁺ T cell lymphocytosis in the periphery by 4–5 wk of age (30). CTLA-4 is a member of the CD28 family that negatively regulates T cell activation. Mice homozygous for a targeted deletion in the gene encoding CTLA-4 accumulate T cell blasts expressing markers of activation in several organs, including the liver, heart, and lungs, and become noticeably ill by 2 wk of age and moribund by 4 wk of age (31). Gozalo-Sanmillan et al. (24) used spectratype analysis to compare the TCR repertoire diversity in Jak3^–/– mice and in CTLA-4^–/– mice. T cell populations in Jak3^–/– mice exhibit a skewed distribution of CDR3 lengths, indicating that specific Ag is involved in the activation and expansion of the peripheral T cells. By contrast, CTLA-4^–/– T cell repertoires are diverse and unbiased, consistent with a polyclonal (i.e., specific-Ag-independent) activation of T cells. Thus, both mouse models exhibit striking CD4⁺ T cell peripheral activation and expansion, but achieve these through distinct mechanisms. Notably, the severe phenotype of TGF-1^–/– mice is much more similar to the dramatic inflammatory phenotype of CTLA-4^–/– mice than to the comparatively milder phenotype of Jak3^–/– mice. Hence, the finding that spectratype profiles from TGF-1^–/– mice are highly perturbed (i.e., Jak3^–/–- like) rather than diverse and unbiased (i.e., CTLA-4^–/–- like) was somewhat surprising. This finding shows that TGF-1 and CTLA-4 use distinct mechanisms to regulate T cell expansion and the development of autoimmunity.

Implications for TGF-1 function

The process of negative selection is imperfect, and within the pool of thymic emigrants are T cells with the potential to react with self-Ags. These cells enter the periphery and circulate within secondary lymphoid organs, sampling self-Ags presented by dendritic cells. The interaction of a self-peptide/MHC with a cognate CD4⁺ T cell is typically an abortive, self-limited one. In the absence of TGF-1, however, this encounter triggers the activation, expansion, and effector activity of self-reactive T cells, production of pathologic levels of IFN- and other effector molecules, modulation of parenchymal cells (such as the up-regulation of class II expression (29)), and subsequent end-organ destruction. The processes that result in immune-mediated destruction of the liver appear to begin as early as 3 days after birth and possibly before. This underscores the vital, nonredundant role played by TGF-1 in regulating the response of CD4⁺ T cells to self-Ags presented in the periphery. In support of this, restricting the TCR repertoire available to CD4⁺ T cells through expression of a transgenic TCR prevents the development of inflammation and end-organ damage in TGF-1^–/– mice (9, 10). Along with the current work, these data strongly suggest that the inflammatory pathology in TGF-1^–/– mice results from the interaction of autoreactive CD4⁺ T cells with self-Ags. The Ags that drive the disease process are likely to be derived from self-proteins, rather than from proteins produced by organisms of the intestinal microflora, as TGF-1^–/– mice raised under germfree or standard conditions develop similar inflammatory pathologies (11).

There are several potential mechanisms by which TGF-1 prevents the pathological activation and activity of autoreactive cognate CD4⁺ T cells. Several studies show that CD4⁺CD25⁺ regulatory T cells (Tregs) may use TGF-1, possibly presented on the T cell surface (32) as an important effector molecule to inhibit the proliferation of Th cells (33, 34). Kinetic studies of Treg development show that thymus-derived CD4⁺CD25⁺ T cells become detectable in the periphery of BALB/c mice beginning at day 4, but not before (35). Because BALB/c-TGF-1^–/– mice exhibit TCR repertoire skewing as early as day 3, it seems unlikely that a defect in the Treg immune response entirely accounts for the CD4⁺ TCR abnormalities observed. In support of this, the development and function of Treg cells appear to be intact in TGF-1^–/– mice (33, 36). Thus, TGF-1 likely has a critical role in regulating conventional T cells distinct from its role in the Treg system. Consistent with this hypothesis, Li et al. (37) have shown recently that mice with a conditional deletion of the TGF-1 receptor in the T cell compartment exhibit spontaneous T cell-mediated autoimmunity, and that TGF-1 regulates effector T cells through pathways that are independent of Treg cells. TGF-1^–/– thymocytes have a lowered threshold for TCR activation, responding strongly to TCR signals that are otherwise insignificant to wild-type thymocytes (38). At the molecular level, TGF-1 inhibits proximal TCR signaling, as well as the Tec kinase ITK, Ca²⁺ flux, and nuclear translocation of NFAT (39). The adaptor molecule cbl-b appears to be another key target of TGF-1 in modulating the response of T cells to signaling through the TCR (40). Thus, through modulating TCR signaling, TGF-1 may determine whether the encounter of autoreactive T cells with self-Ag results in a benign or malignant T cell response.

In addition to its effects on TCR signaling, TGF-1 also shapes the differentiation of Th cells in the periphery. TGF-1 inhibits Th1 differentiation through the inhibition of the expression of the transcription factor T-bet (41, 42), the expression of which is both necessary and sufficient for Th1 development (43). We have shown recently that TGF-1 requires the protein tyrosine phosphatase Shp-1 to inhibit IFN--mediated up-regulation of T-bet expression (44). TGF-1 is capable of recruiting CD4⁺CD25^– T cells to the Treg effector pool, via SMAD3-mediated up-regulation of the key Treg transcription factor FoxP3. However, as mentioned, TGF-^–/– mice have intact Treg numbers and function, so the relevance of this aspect of TGF-1 function to its control of autoimmune liver disease is not obvious. Recently, several groups have shown that TGF-1, in conjunction with IL-6, can elicit the development of a distinct lineage of effector CD4⁺ T cells producing the inflammatory cytokine IL-17, but not IFN- (45, 46). Hepatocellular damage in TGF-1^–/– mice is dependent upon IFN-, but a specific requirement for IL-17 has not yet been tested. Microarray analyses of TGF-1^–/– liver RNA indicate high levels of IFN- mRNA, but normal levels of IL-17 mRNA (R. T. Robinson and J. D. Gorham, unpublished data), and a specific role for IL-17 in autoimmune liver disease remains to be tested.

	Acknowledgments

 
We thank Brent Berwin (Dartmouth Medical School) and Ed Usherwood for critically evaluating the manuscript. We are indebted to Christine Kretowicz and Beverly Gorham for expert breeding and screening of TGF-1^–/– mice and TGF-1^+/– mice.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI053056 and DK073904 and P20RR16437 from the COBRE Program of the National Center for Research Resources.

² Address correspondence and reprint requests to Dr. James D. Gorham, Department of Pathology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756. E-mail address: James.D.Gorham{at}Dartmouth.edu

³ Abbreviations used in this paper: CD62L, CD62 ligand; Treg, regulatory T cells; CD4SP, CD4⁺ single positive; MNC, mononuclear cell; ALT, alanine aminotransferase; SC, spectratype complexity.

Received for publication January 22, 2007. Accepted for publication April 19, 2007.

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