Accelerated transition from the double-positive to single-positive thymocytes in G
i2-deficient mice
Yujin Zhang1,
Milton J. Finegold1,
YongZhu Jin2 and
Mei X. Wu1,2
1 Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA
2 Wellman Center of Photomedicine, Massachusetts General Hospital and Department of Dermatology, Harvard Medical School, Boston, MA 02114, USA
Correspondence to: M. X. Wu; E-mail: mwu2{at}partners.org
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Abstract
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Deletion of
i2 subunit of heterotrimeric G proteins induces a 24-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G
i2 is involved in thymocyte development is unknown. To determine a role for G
i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G
i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G
i2. Lack of G
i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G
i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vß gene, aberrant development of CD4+CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G
i2 protein in clonal selection and functionality of thymocytes.
Keywords: colitis, Gai2, positive selection, TCR
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Introduction
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Heterotrimeric G proteins consist of
, ß and
subunits, and they are GTP-binding proteins that participate in intracellular signal transduction by coupling with seven transmembrane receptors with which an array of hormones and chemokines interact (13). G
i proteins, including G
i1, G
i2 and G
i3 in this family, are involved in the inhibition of adenyl cyclase, activation of phosphoinositide 3 kinase and phospholipase C (PLC) and regulation of Ca2+ mobilization (47). All three Gi proteins can be uncoupled from receptors by pertussis toxin (PTX), a potent Th1 adjuvant produced by Bordetella pertussis (2, 810). Mice with gene-targeted deletion of the
i2 subunit (G
i2/) developed chronic colitis, manifested by wasting, diarrhea and bloody stools, that clinically closely resemble ulcerative colitis, but immunologically, it is similar to Crohn's disease (11). The mice had a local increase in memory T cells, in pro-inflammatory Th1-type cytokines and in infiltration of activated CD4+ T cells in the intestinal mucosa (12). Consistent with this, peripheral T cells isolated from healthy G
i2/ mice, including splenic and lymph node T cells and perhaps thymocytes, all displayed a hyperimmune response, as is shown by enhanced proliferation and production of inflammatory cytokines when stimulated with various mitogens, suggesting that G
i2 protein negatively regulates T cell immune responses in general (1113).
In addition to a hyperimmune response in the gut, G
i2-deficient mice had a 24-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes when compared with wild-type littermates, which occurs prior to colitis development (11), indicating an indispensable role for G
i2 in thymocyte development. Yet, how G
i2 protein influences thymocyte differentiation is unknown at present. G
i2 seems to play a role in thymocyte development at stages only after the formation of the double-positive (DP) thymocytes because the proportions of the DP (CD4+CD8+) and double-negative (DN, CD4CD8) thymocytes are comparable in the presence versus absence of G
i2 (11). Maturation from the DP to SP thymocytes involves clonal selection that represents a hallmark of the thymus and is an essential mechanism for generating a unique T cell repertoire, avoiding autoimmunity. Clonal selection including positive and negative selection is governed by interaction of the TCRs with self-peptide/self-MHC complexes (14, 15). Thymocytes that express TCR capable of interacting with self-MHC molecules at low affinity are positively selected and mature into the SP thymocytes, whereas thymocytes expressing TCRs that cannot interact with MHC molecules with sufficient affinity fail to undergo positive selection and die by neglect. Thymocytes with TCR expression at a high frequency or a high affinity are negatively selected. Although the TCRs and MHC molecules directly control clonal selection, other downstream molecules can strengthen or weaken the signal transmitted by the TCRs, for instance, ras, raf, mitogen-activated protein kinase, Ca2+, and protein kinase C (PKC) (1619), thus having a great impact on the outcome of T cell maturation (20).
In the present study, we demonstrate that lack of G
i2 protein accelerates the transition from DP to SP thymocytes, through an MHC molecule-dependent manner. Deletion of G
i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The altered response of G
i2/ thymocytes to TCR engagement promotes positive selection and may also rescue some thymocytes from death caused by neglect due to an insufficient affinity of the TCR they express. The abnormality resulted in a high number of the SP thymocytes including CD4+CD25+ T regulatory (Treg) cells in the mice at early age, and an altered usage of TCR Vß gene with increased expression of Vß8.1/2, Vß12 and Vß13, but decreased expression of Vß4 and Vß8.3. When the mice aged, they displayed untimely thymic involution, due at least in part to rapid exhaustion of the DP thymocytes. These findings reveal for the first time an important role for G
i2 protein at clonal selection in the thymus.
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Methods
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Animals
G
i2-deficient mice (129Sv/C57 background) were generated by gene targeting and backbred with C57BL/6 (B6) mice for five to six times as described (11, 12). ß2-microglobulin (ß2m)//I-Aßb/ double knockout (ko) mice were purchased from Taconic Farms (Germantown, NY, USA) and they were cross-bred with G
i2/ mice to generate MHC/G
i2-deficient mice. The genotypes at the ß2m and I-Aßb loci were determined by PCR using the following primers. For ß2m locus detection, the forward primer is 5'-AAACTGCAGTCATCTTCCCCTGTGGCCCTCAGA-3' and the reverse primer is 5'-GTAAGGAAGAACTTGAGGCTTACC-3'. For I-Aßb locus detection, the forward primer is 5'-AGCACCGCGCGGTGACCGAG-3' and the reverse primer is 5'-CAGAGGGCAGAGGTGAGACAG-3'. Lack of a corresponding PCR product was identified as homozygous disruption in each case. The mice were housed in conventional cages at the animal facilities of the Baylor College of Medicine or Massachusetts General Hospital in accordance with the institute's guidelines.
Flow cytometry
Single-cell suspensions were prepared from the thymus or spleen at the indicated age and stained with a mixture of biotinylated and/or fluorochrome-conjugated antibodies. The antibodies including FITC-conjugated anti-CD4, anti-CD8, anti-Vß8 and anti-CD3; PE-conjugated anti-CD8 and PE-conjugated anti-CD4; biotin-conjugated anti-CD3, anti-CD25 and anti-CD8 and cy-Chrome (CyC)-conjugated streptavidin were all purchased from BD PharMingen (San Diego, CA, USA). The stained cells were analyzed by a FACScan or a FACSCalibur (Becton-Dickinson) cytometer equipped with Cellquest software (San Josa, CA, USA). For analysis of TCR repertoire, thymocytes were stained with a panel of mAbs recognizing TCR Vß chain 2, 3, 4, 5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10b, 11, 12, 13, 14 and 17a (a Mouse Vß TCR screening kit, PharMingen) as per the manufacturer's instructions.
Study of cell division using the vital dye carboxyfluorescein diacetate succinimidyl ester
Thymocytes isolated from 1- to 2-day-old newborn mice were stained with PEanti-CD4 antibody and biotinanti-CD8 antibody plus CyC-conjugated streptavidin and DP thymocytes were sorted on a Beckman-Coulter Altra high-speed sorter. The resulting T cells had a purity of >99% and were incubated at 37°C for 15 min with the vital dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes Eugene, OR, USA) at a final concentration 10 µM in complete culture medium consisting of RPMI 1640, 10% FCS, 2 mM L-glutamine, 25 mM HEPES buffer, 50 µM ß-mercaptoethanol, 100 U/ml of penicillin and 100 µg/ml of streptomycin. After two washes with serum-free medium, the DP thymocytes at a density of 3 x 106 were re-suspended in complete culture medium, seeded to 24-well plates that were pre-coated with 10 µg/ml of anti-CD3 antibody (2C11) and incubated at 37°C. The cells were also stimulated with Con A at a final concentration of 4 µg/ml or phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) plus ionomycin (1 µM). The cells were harvested 4 days after the stimulation, fixed with 1% formaldehyde in PBS on ice for 10 min, washed and analyzed by flow cytometry.
Thymocyte emigration
Mice were anesthetized with ketamine/xylazine (100/10 mg/ml, 10 µl per mouse) intramuscularly. A mediastinal incision was made to expose the thymus. Ten microliters of CFSE (200 µM) was administered into each thymic lobe and the skin overlying the chest wall was then closed with surgical clips. Control mice received the same volumes of PBS with dimethyl sulfoxide at the appropriate dilution. At various times following intra-thymic injection, CFSE-labeled T cells in the lymph nodes, including axillary and inguinal lymph nodes, were detected by flow cytometry after staining with PE-conjugated anti-CD3 as described.
Cell cycle analysis
To isolate CD4 SP thymocytes for cell cycle analysis, total thymocytes were stained with PEanti-CD4 and FITCanti-CD8 antibodies as described. CD4 SP thymocytes were sorted on a Beckman-Coulter Altra high-speed sorter. DNA content of the purified CD4 SP thymocytes was stained with propidium iodide (Calbiochem, San Diego, CA, USA) using a standard protocol. In brief, the cells were fixed in 70% ethanol for 1 h, washed and then incubated for 30 min in staining buffer containing propidium iodide at 50 µg/ml, RNase A at 0.2 mg/ml and 0.1% Nonidet P-40, followed by analysis on FACScan.
Thymocyte apoptosis
Thymocytes (2 x 106) were plated in 24-well plates and treated with immobilized anti-CD3 antibody (10 µg/ml1), dexamethasone (0.01 M) or PMA (100 ng/ml1). One day later, apoptotic cells were determined by staining with PE-conjugated anti-CD4, biotin-conjugated CD8 plus CyC-conjugated streptavidin and FITC-conjugated anti-Annexin V, followed by flow cytometric analysis.
Statistical analysis
The Student's two-tailed t-test was used to analyze the significance between experimental groups and relevant controls.
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Results
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Similar thymic emigration in the presence versus absence of G
i2
A 24-fold increase in the proportion of the SP thymocytes in G
i2-deficient mice recapitulates the phenotype obtained in transgenic mice expressing PTX S1 subunit in T cells under the control of lck promoter (21). This, in line with a recent finding that lack of the Gi protein-coupled receptor CCR7 abrogated emigration of newly generated mature T cells (22), raised a possibility that G
i2 deficiency impaired emigration of mature thymocytes. We therefore set out to assess a role for G
i2 protein in thymic emigration by counting neonatal T cells in the spleens of newborn mice daily up to 5 days after birth (22). To circumvent the difficulty that homozygous female mice have a relatively low birth rate due to illness in their early life, heterozygous (+/) female and homozygous (/) male mice were bred, which would theoretically yield similar numbers of homozygous (/) and heterozygous (+/) mice. Moreover, no significant difference in thymocyte development was observed between heterozygous and wild-type mice (our unpublished observation) (11). Heterozygous littermates thus served as controls in place of wild-type mice in the present study unless otherwise indicated, to minimize the variations from the parental mice. As shown in Fig. 1(A, left panel), few T cells were detected in the spleen of newborn mice at day 0 in both ko and control mice. Yet, the number of T cells in the spleen increased dramatically within days, representing recent thymic emigrants (22). Superficially, neonatal T cells in the spleen of G
i2-deficient mice appeared to be relatively fewer in number than those in control mice, in particular, at days 45. However, after normalizing for the decreased body mass of the G
i2/ mice which is
20% less than that of their control littermates (11), the total number of neonatal splenic T cells was actually constant between +/ and / G
i2 mice. A decrease in the number of T cells in the neonatal spleen due to the smaller body size of G
i2/ mice is well illustrated by comparable decreases in the number of thymocytes in parallel experiments, in spite of a 24-fold increase in the proportion of the SP thymocytes (Fig. 1A, right panel). Furthermore, following intra-thymic administration of CFSE vital dye, the proportion of CFSE+/CD3+ T cells rose linearly from day 1 to day 7 in the lymph nodes in wild-type mice (Fig. 1B), representative of thymic emigrants (23). A similar increase in the proportion of CFSE+/CD3+ T cells was also observed in the absence of G
i2. Apparently, these results did not support a role for G
i2 protein in thymic emigration.
Accelerated transition from the DP to SP stage in G
i2/ mice
To investigate whether differentiation of the DP to SP T cells was altered in lack of G
i2, we studied thymocyte ontogeny after 14 days of gestation, as bone marrow-derived precursor cells (pre-T cells) seed the thymic rudiment between days 10 and 13 of gestation (24). We found no significant alteration in the subsets of fetal thymocytes including DN, DP and SP CD4 or CD8 cells at 14, 18 or 20 days of gestation in the presence versus absence of G
i2 protein (data not shown). A slightly higher level of CD8 SP T cells was seen 1 day after birth in G
i2/ compared with G
i2+/ mice (14.8 versus 12.4%), but lacking statistical significance (data not shown). At day 2, the percentages of DN, DP and SP CD4 T cells were still similar in G
i2/ mice and control littermates, except that the proportion of CD8 SP T cells remained higher (13 versus 9%) in G
i2/ mice (Fig. 2A). Starting at day 3, a decrease in CD8 SP T cells and a concurrent increase in the number of CD4 SP cells took place in both mice, but to a greater degree in G
i2/ mice. Remarkably, at day 4,
18% of G
i2/ thymocytes were CD4 SP T cells, an
3-fold increase over the 6% of CD4 SP T cells obtained in control mice (Fig. 2A). The increased proportion reflected an increased production of CD4 SP T cells, as the absolute number of CD4 SP thymocytes was increased by 2.5-fold relative to control mice (53 ± 5 x 105 versus 21 ± 2 x 105) (Table 1). Notably, a semi-mature sub-population of CD4 SP thymocytes was sometimes presented in 4-day-old mice, but not in 7-day-old G
i2/ mice, which expressed intermediate levels of CD4. Accompanying an increase in the percentage of the SP thymocytes was a concomitant decrease both in the percentage and in the absolute number of the DP thymocytes (Fig. 2A, Table 1). The percentages of the DP thymocytes were reduced from 86% in control mice to 61% in G
i2/ mice, and the absolute numbers were diminished by 67% (3 x 107 versus 1.8 x 107) (Table 1), suggesting exaggerated transition from the DP to SP thymocytes in the lacking of G
i2.

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Fig. 2. Ontogeny of thymocytes in newborn mice. Thymocytes were isolated from G i2/ mice and control littermates as in Fig. 1 at the indicated days (A) or weeks (wks, B). Thymocytes were fixed, stained with PEanti-CD4 and FITCanti-CD8 antibodies and analyzed by flow cytometry. The numbers within panels indicate the relative percentages of positively stained cells in the gated sample. One representative result of five experiments performed is shown.
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An increase in production of the SP thymocytes without a concurrent increase in the total number of thymocytes in G
i2/ mice was inconsistent with expansion of the SP thymocytes after clonal selection. Moreover, the cell cycle analysis of the SP thymocytes showed comparable percentages of cells that were in S + G2M phase between G
i2/ and control littermate (data not shown), ruling out that increased production of the SP thymocytes results from proliferation of selective population of thymocytes in post-selection. In addition, the drastic increase in CD4 SP thymocytes at day 4 did not concur with a decrease in emigration of the SP T cells into the spleen and lymph nodes (Fig. 1A and B), in which thymic emigration to the spleens or lymph nodes was indistinguishable between these two groups of mice. Lastly, to rule out that the decreased number of the DP thymocytes was attributable to increased apoptosis, we analyzed apoptosis susceptibility in thymocytes isolated from G
i2/ mice. When thymocytes were treated with immobilized anti-CD3 antibody, PMA + anti-CD3 antibody, dexamethasone or serum starvation, followed by Annexin flow cytometry, no significant difference in apoptosis of thymocytes was found regardless of whether they were isolated from wild-type, heterozygous mice or G
i2/ mice (data not shown). The observation is consistent with previous investigation showing that lack of G
i2 did not facilitate apoptosis in thymocytes (11). Moreover, we also investigated whether the absence of G
i2 affected apoptosis in thymocytes only during the DP to SP transition stage, which might be overlooked when a whole thymocyte population was studied, by staining of thymocytes with FITCanti-Annexin V, biotinanti-CD8 plus CyC-conjugated streptavidin and PEanti-CD4. Percentages of Annexin V-positive cells were then compared on gated subsets of CD4+CD8+ DP, CD4dimCD8dim, CD4brightCD8dim and CD4dimCD8bright T cells. Once again, we failed to observe a significant increase in the percentages of apoptotic cells in the absence versus presence of G
i2 protein (data not shown). Similar susceptibility to undergo apoptosis in the presence versus absence of G
i2 thus makes it implausible that shrinking in the number of the DP thymocytes is ascribed to accelerated apoptosis of the DP thymocytes. Taken together, these studies reinforce the conclusion that expedited transition of the DP to SP thymocytes is the reason for increased production of the SP thymocytes in G
i2/ mice.
As the mice aged, accelerated transition of the DP to SP thymocytes continued at a similar pace and resulted in severe depletion of the DP thymocytes. As can be seen in Fig. 2(B) and Table 1, the percentages of the DP thymocytes declined drastically over 2 months of age, from 34% at 3 weeks to 19% at 6 weeks and to 5% at 8 weeks of age, commensurate with a decrease in total numbers of thymocytes from 1.5 x 108 at 3 weeks to 6 x 107 at 5 weeks and to 2.4 x 106 at 8 weeks of age. Therefore, despite a continuous rise in the proportions of CD4+ and CD8+ mature T cells in the thymus, the absolute numbers of the SP thymocytes were shrinking when the mice were approaching 2 months of age. In contrast, the percentage of the DP thymocytes remained relatively constant in control mice, in spite of a slow but steady decrease in total numbers of thymocytes. Exhaustion of the principle source of the DP thymocytes is probably one of the primary reasons for a decrease in the production of the SP thymocytes. However, the possibility of thymic involution associated with onset of autoimmunity could not be ruled out, although no overt signs of inflammation in the mice were noticed at the time of experiment.
An enhanced response to TCR ligation in G
i2/ thymocytes
Maturation from DP to SP thymocytes is driven by interaction of the TCRs with appropriate self-MHC/self-peptide complexes that determines the fate of maturing thymocytes. Deletion of G
i2 may augment the overall amplitude and/or quality of the signals transmitted by the TCRs, and consequently the transition of the DP to SP thymocytes, because previous studies have shown enhanced thymocyte proliferation following engagement of the TCR/CD3 complex in G
i2-deficient mice (11). However, it is unclear whether or not the enhanced proliferation of thymocytes in their studies is attributed to a greater portion of the SP T cells in thymocytes isolated from G
i2-deficient mice, as the study utilized unfractionated thymocytes from 6-week-old mice. This is an age marked by a drastic increase in the proportion (>70%) of the SP T cells that is almost 3-fold greater than the 25% of the SP thymocytes normally seen in control mice (Fig. 2B, Table 1). SP thymocytes are much more potent to stimulation with ligation of the TCR/CD3 complex. To circumvent these confounding factors, we used 1- to 2-day old neonatal thymocytes, whose ontogeny is developmentally indistinguishable between G
i2/ and control mice (Fig. 2A). Additionally, DP thymocytes, instead of unfractionated thymocytes, were used for the proliferation study. The DP thymocytes were labeled with CFSE vital dye and stimulated by ligation with the CD3/TCR complex. During proliferation of CFSE-stained cells, the dye is halved with each consecutive cell division so that cell proliferation can be determined from stepwise decreases in fluorescence intensity. As can be seen in Fig. 3, thymocytes from G
i2+/ and G
i2/ mice were all maximally labeled on day 0. By day 4, a small number (<5%) of control thymocytes underwent more than one cell division, which was however increased to 37% in the absence of G
i2; a 9-fold increase. Moreover, the maximal number of cell divisions after a 4-day stimulation was increased from two in wild-type thymocytes to six in G
i2/ thymocytes, as deduced by the CFSE intensity decreasing from 2000 to 500 in the former but to
30 in the latter. A hyper-proliferation was even more drastic after Con A stimulation (61 versus 5%) (Fig. 3), clearly indicating that thymocyte activation following ligation of the TCRs is much stronger in the absence of G
i2. Unlike stimulation with Con A or anti-CD3 antibody, PMA plus ionomycin that bypasses the TCRs stimulated vigorous proliferation in thymocytes even in the presence of G
i2, which could not be advanced by null mutation of
i2 gene, an observation similar to that described in peripheral T cells (13). The results implicate that null mutation of the G
i2 gene strengthens TCR-mediated signaling by regulating the activities of PKC and/or Ca2+ mobilization, which can directly affect thymic positive selection (25).

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Fig. 3. Enhanced proliferation of thymocytes in the absence of G i2. DP thymocytes isolated from 1- to 2-day old pups were labeled with CFSE and stimulated for 1 h (D0) or 4 days (D4) with immobilized anti-CD3 antibody (10 µg/ml), 4 µg/ml of Con A or PMA (50 ng/ml) + ionomycin (1 µM), followed by flow cytometry. The relative percentages of cells within each panel are indicated, and the experiment was repeated with similar results for a total of three times.
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Effects of G
i2 deficiency on thymocyte differentiation dependent on MHC molecules
If the altered transition from the DP to SP thymocytes is attributable to a strengthening of a signal of the TCRs or a lowering the threshold of thymocyte activation in G
i2/ mice, it must require interaction between the TCRs and self-peptide/self-MHC molecule complexes. To test this, we crossed G
i2/ mice with ß2m//I-Aßb/ mice which lack both MHC class I and II molecules (26). As shown in Fig. 4, MHC-deficient mice had a few CD8 (0.8%) and CD4 (2.2%) SP T cells that were essentially unaffected while the G
i2 gene was null mutated. Of note, the percentage of CD8 SP T cells, though at a very low level, was increased by 3-fold in MHC-deficient mice in the presence relative to the absence of G
i2 (0.8 versus 2.4%). This trend was consistent in all ß2m/ mice, probably due to the fact that the mice express a low level of MHC class I molecule (27). Our studies showed that the effects of G
i2 deficiency required expression of MHC class II for CD4 or class I for CD8 SP cell development. Thus, in class II/G
i2/ double ko mice, the percentage of CD8 SP cells was increased by almost 2-fold, but the percentage of CD4 SP cells remained little changed, when compared with class II/G
i2+/ mice. Likewise, a 2-fold increase in the maturation of CD4 SP cells was clearly seen in the presence of MHC class II, but in the absence of both MHC class I and G
i2 molecules. A loss of function in the absence of MHC molecules suggests that G
i2 deficiency affects differentiation of CD4 and CD8 SP thymocytes only after the TCRs have made contact with peptides presented by either MHC class I or MHC class II molecule. G
i2 has no effect on the lineage commitment of CD4 and CD8 SP cells.

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Fig. 4. Enhanced maturation of the SP thymocyte depends on an appropriate MHC molecule. CD4 and CD8 expression patterns of thymocytes from progeny of the G i2/ mice cross-bred with MHC/ mice were analyzed at 1014 days after birth by flow cytometry. Numbers in each profile indicate percentages of cells in corresponding quadrants with a total of 50 000 events analyzed for each sample. Similar results were obtained from three independent experiments.
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Alterations in TCR Vß gene usages and in the development of CD4+CD25+ Treg cells
Increased production of the SP thymocytes might alter a TCR repertoire and allow some DP thymocytes to be falsely selected in G
i2/ mice. To test this, we evaluated TCR Vß chain usages in CD4 SP thymocytes that are critical in colitis development in G
i2 mice (12, 28, 29). Compared with those found in control mice, significant increases in the percentages of T cells expressing Vß8.1/2, 12 and 13 but decreases in Vß4 and 8.3 were observed in G
i2/ mice (Fig. 5A), suggesting a possible alteration in the TCR repertoire in the mouse.

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Fig. 5. Alterations in TCR Vß gene usages and CD4+CD25+ Treg cells. Thymocytes were prepared from G i2/ mice and indicated control mice at 34 weeks of age. The cells were stained with anti-CD4 and anti-CD8 antibodies and a panel of mAbs recognizing TCR Vß chain (A) or anti-CD25 antibody (B), and analyzed by flow cytometry. (A) Analysis of CD4+CD8 thymocytes by a panel of 17 anti-Vß mAbs. Statistical differences were determined using the t-test; *P < 0.05, **P < 0.01 and n = 6. (B) CD4+CD25+ Treg cells in gated CD4+CD8 thymocytes. Percentages of CD4+CD25+ thymocytes relative to the total number of CD4 SP cells are indicated in each histogram. Representative results of eight mice analyzed in each group are shown (P < 0.05).
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In addition, thymic-derived dysregulated tolerance has been recently suggested to be a key event in inflammatory bowel disease (IBD) development (30, 31). The increased transition of the DP to SP thymocytes due to an altered TCR signaling raises a distinct possibility that development of CD4+CD25+ Treg may be impaired in this mouse, since TCR signaling is required for their selection (32, 33). As expected, when CD25 expression was analyzed in gated CD4+CD8 thymocytes, percentages of CD4+CD25+ T cells were significantly enhanced in G
i2/deficient mice compared with wild-type control mice (P < 0.05) (Fig. 5B). This enhancement, in line with increased generation of CD4 SP cells, gave rise to a 2-fold increase in the total number of CD4+CD25+ thymocytes in G
i2/ mice (3.0 ± 0.4 x 106 versus 1.5 ± 0.2 x 106). A similar increase was, however, not observed in the spleen in the absence of G
i2 (data not shown). The disproportional thymic export of Treg cells resembles what has been described for CD4 and CD8 SP thymocytes which, though developing at abnormally high levels in the thymus, are essentially unaltered either in their numbers or in their percentages in the spleen of G
i2/ mice when compared with wild-type control mice, implicating that thymic egress of Treg is regulated in a manner similar to that of the SP thymocytes in the mouse (11).
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Discussion
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Maturation from DP to SP thymocytes is crucial for T cells to acquire capacity of selfnon-self discrimination, to be steadily replenished to the naive T cell pools in the periphery and to ensure the functionality of the T cell repertoire. The maturation is governed by interaction of TCRs with an array of self-peptides in association with MHC molecules in the thymus, and it proceeds at a relatively steady pace throughout life. In the first 3 weeks after birth, when thymocytes are rapidly produced to match the animal's increase in body size, production of the SP thymocytes is increased accordingly, but the proportion of the DP thymocytes transiting into SP thymocytes remains constant. The rate-limited differentiation of the SP thymocytes warrants a wise use of the principle source of the DP thymocytes for a need of a constant output of thymocytes into the periphery throughout life. Our studies show that absence of G
i2 expedites the transition from the DP to SP at day 4 after birth, a trend that was not slowed down until the source of the DP thymocytes wore out. A continuous decline in the number of the SP thymocytes as a result of rapid exhaustion of the DP thymocytes could drive peripheral expansion of T cells in the periphery, known as homeostatic proliferation (34, 35). In accordance with this, healthy G
i2-deficient mice displayed an increased frequency of memory CD4+ and CD8+ cells that were characterized by increased levels of CD44 and decreased levels of CD45RB and CD62L, before clinical signs of disease were evident (36). Conceivably, compensatory expansion of existing T cell pools could give rise to a T cell repertoire skewed toward those against normal enteric flora in the intestines, thereby contributing to spontaneous development of colitis in the mice. This possibility is favored by a recent study showing that reduced T cell number and the resulting exaggerated homeostatic proliferation of T cells can cause autoimmunity (37).
In addition to peripheral expansion, accelerated transition of the DP to SP thymocytes may alter positive selection and/or Treg cell development. Some harmful auto-reactive T cells may escape from clone selection, mature, exit the thymus and react to antigens presented on normal enteric flora, which may also predispose autoimmune responses in the gut lining in G
i2-deficient mice (29, 38, 39). Although a causal relationship between the TCR repertoire and subsequent development of colitis in the mice remains to be established, several lines of evidence have suggested that IBD is associated strongly with an altered TCR repertoire. First, the onset of IBD in G
i2-deficient mice is highly dependent on mouse strains; 129Sv mice develop severe disease, whereas C57BL/6 mice develop moderate disease. The strain-dependent susceptibility to IBD may be in part ascribed to the involvement of self-MHC molecules and self-peptides they presented on thymic stroma. Second, TCR
-, TCRß- or TCRß
-deficient mice and HLA-B27 transgenic rats all had spontaneous chronic or acute IBD, pointing to a direct relationship of TCRs with an onset of the disease (40, 41). Third, IBD was not observed when the mutant mice were maintained in a germ-free environment, but it did occur after reconstitution with a pathogen-free flora (42), suggesting that antigens present on non-pathogenic flora are a primary factor in the initiation of the disease. Nevertheless, it should be pointed out that IBD is a multi-factorial disease and colitis development in G
i2/ mice may involve multiple defects, as has been described in humans. In this regard, the role of Treg cells in predisposing to development of IBD remains to be determined, since no alteration in the numbers of CD4+CD25+ Treg cells was seen in the periphery of the mice at 34 weeks of age, in spite of a 2-fold increase in their numbers in the thymus. Besides the abnormality in T cells, G
i2 deficiency impeded development of IL-10-producing B cells and it was postulated that attenuation of IL-10 production in the mucosal environment might contribute to the hyperimmune response skewed to Th1 (43).
Involvement of G
i2 in thymocyte differentiation at a transition stage from the DP to SP thymocytes is in agreement with our observation that impairment of thymocyte differentiation in G
i2/ mice required the presence of MHC expression. In the absence of MHC molecules, G
i2 shows no effects on thymocyte differentiation. In the presence of a low level of MHC class I expression like ß2m/ mice (27), G
i2 deficiency can still enhance the maturation of CD8 SP T cells proportionally. This raises the possibility that G
i2 deficiency affects only thymocytes expressing TCRs that interact with peptide/MHC molecular complexes at a low affinity or a low frequency, probably by increasing the overall amplitude and/or quality of a relatively weak signal transmitted by the TCRs or lowering the threshold for thymocyte activation triggered by ligation of the TCR/CD3 complex. Thymocytes expressing the TCRs at a high frequency or a high affinity may be committed to apoptosis (negative selection) at upstream molecules before G
i2-signaling and TCR-mediated signaling pathways converge.
Alternatively, G
i2 deficiency may impair chemorepulsion or cell migration away from a stimulus so that the duration of the interaction between the TCRs and peptide/MHC complexes would be extended on the cortical epithelial cells, where positive selection occurs. The longevity of interaction of TCRs and MHC molecules would certainly have an impact on the transition of the DP to SP thymocytes. In this regard, a body of evidence indicates that PTX-sensitive Gi protein-coupled receptors, including CXCR4, CCR7, CCR9 and CCR4, play a decisive role in thymocyte development (4448). CXCR4 receptor is highly expressed on immature DN and DP thymocytes and the corresponding ligand CXCL12 (also called stromal cell-derived factor-1) is produced by thymic epithelial cells in the sub-capsular and medullary regions. The chemokine preferentially attracts immature DN and DP thymocytes across the corticomedullary junction in the thymus (44, 49, 50). During positive selection, CCR7 is induced on thymocytes (48, 49) and it can bind to the medullary chemokines CCL19 and CCL21, providing guidance for the developing thymocytes moving from the cortex to the medulla in the thymus (44, 51). Another CC chemokine CCL22 (macrophage-derived chemokine) that is secreted by macrophages in the medullary region selectively directs transitional thymocytes between the DP and SP stages out of the cortex by binding their receptor CCR4 (48). Despite an essential role for chemokines in thymocyte development, deletion of individual chemokine receptor or its ligand fails to generate a phenotype similar to that observed in G
i2/ mice (45, 52). Therefore, G
i2 protein must be coupled to more than one of these chemokine receptors in order to regulate thymocyte differentiation.
It is clear that interaction of the TCRs with MHC/peptide molecules directs the transition from the DP to SP stage. However, other downstream molecules can contribute to the overall amplitude and/or quality of signals transmitted by the TCRs, having an impact on the outcome of clonal selection in thymocyte development (17). For instance, deletion of the Thy gene shows augmented TCR signaling and impairs maturation from the DP to SP stage of thymocyte development (53). In contrast to G
i2/ mice, Thy/ mice exhibited a 23-fold decrease in the proportion of the SP thymocytes, due to inappropriate negative selection. The src family protein tyrosine kinase p56lck can enhance signals mediated through the TCR/CD3 complex (54). Over-expression of a dominant-negative p56lck inhibits positive selection and consequently reduces the numbers of the SP thymocytes when expressed under the control of the distal promoter. The ability of PMA plus ionomycin to override the effect of G
i2 deficiency on thymocyte proliferation suggests that absence of G
i2 may strengthen the activation signal of the TCRs upstream of or at Ca2+-dependent PKC. A similar hyper-proliferation was also described recently in peripheral T cells in G
i2/ mice when stimulated with ligation of the TCR/CD3 complex (13). These findings conclude a general role for G
i2 in the down-regulation of T cell proliferation.
G
i proteins are well known for their roles in leukocyte homing, which can be abolished by treatment with PTX. The roles of G
i proteins in regulation of proliferation of T cells are revealed only after G
i2-deficient mice were made, emphasizing the value of null mutation of individual G
i genes. The current investigation shows that G
i2 is critical for the control of a transition rate of the DP thymocytes differentiating into the SP thymocytes in the thymus, ascribing a previously unappreciated function to the multiple roles of G
i2 in regulating immune responses.
 |
Acknowledgements
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We thank members of the Texas Gulf Coast Digestive Diseases Center for stimulating discussion, Lu Zhang for technical assistance and animal husbandry and R. Edwards for critical reading of the manuscript. This work is supported in part by the National Institutes of Health (NIH) grant AI050822, the Research Scholar Grant RSG-01-178-01-MGO from the American Cancer Society, a Moran foundation award (PRJ 00-114) from the Baylor College of Medicine (to M.X.W.), the NIH project grant DK43351 (to D. K. Podolsky) and the Public Health Service grant DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.
 |
Abbreviations
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---|
CFSE | carboxyfluorescein diacetate succinimidyl ester |
CyC | cy-Chrome |
DN | double negative |
DP | double positive |
IBD | inflammatory bowel disease |
ko | knockout |
ß2m | ß2-microglobulin |
PKC | protein kinase C |
PLC | phospholipase C |
PMA | phorbol-12-myristate-13-acetate |
PTX | pertussis toxin |
SP | single positive |
Treg | T regulatory |
 |
Notes
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Transmitting editor: T. Tedder.
Received 5 August 2004,
accepted 6 December 2004.
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