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The Journal of Immunology, 2001, 167: 6270-6278.
Copyright © 2001 by The American Association of Immunologists

Requirement for Ca2+/Calmodulin-Dependent Kinase Type IV/Gr in Setting the Thymocyte Selection Threshold1

Vidya Raman*, Frank Blaeser*, Nga Ho*, Deborah L. Engle*, Calvin B. Williams* and Talal A. Chatila2,*,{dagger},{ddagger}

Departments of * Pediatrics and {dagger} Pathology and Immunology and {ddagger} Center for Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The outcome of thymocyte selection is influenced by the nature of Ca2+ signals transduced by the TCR. Robust Ca2+ responses characterize high-affinity, negatively selecting peptide/TCR interactions, while modest responses typify lower-affinity, positively selecting interactions. To elucidate mechanisms by which thymocytes decode distinct Ca2+ signals, we examined selection events in mice lacking Ca2+/calmodulin-dependent protein kinase type IV/Gr (CaMKIV/Gr), which is enriched in thymocytes. CaMKIV/Gr-deficient thymocytes exhibited impaired positive selection and defective Ca2+-dependent gene transcription. Significantly, CaMKIV/Gr deficiency raised the selection threshold of peptide/TCR interactions such that a peptide that normally induced weak negative selection instead promoted positive selection. These results demonstrate an important role for CaMKIV/Gr in sensitizing thymocytes to selection by low-affinity peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The outcome of thymocyte development hinges on the interaction of TCRs and self peptides presented by MHC class I and II molecules on thymic stromal cells (1). Thymocytes whose TCR fail to recognize peptide/MHC complexes are eliminated by a process of "death by neglect." Thymocytes whose TCR do recognize self peptide/MHC complexes are further selected based on the relative avidity of the overall interaction, which includes the affinity of the relevant peptide ligand/TCR pair, their cell surface density, and the contribution of coreceptor and adhesion molecules (2, 3, 4). Low avidity interactions promote thymocyte survival and differentiation by a process of positive selection. In contrast, thymocytes that interact with higher avidity undergo negative selection by apoptosis, an adaptive process that abrogates the development of autoreactive T cells (4, 5, 6, 7).

The capacity of a thymocyte to differentially respond to positively and negatively selecting ligands engaging the same TCR has focused attention on the nature of signaling events triggered by the respective selection program (8). Of these, the influx of extracellular Ca2+ following TCR engagement has been implicated in influencing the outcome of both positive and negative selection (9, 10, 11). Different patterns of Ca2+ influx are triggered by positively and negatively selecting peptides. High-affinity interactions between the TCR and peptide/MHC complexes provoke robust Ca2+ mobilization, while low-affinity interactions result in modest Ca2+ responses (11, 12, 13). The qualitatively distinct Ca2+ signals delivered by positively and negatively selecting ligands may promote different selection outcomes by activating distinct subsets of intracellular signaling pathways, as has been demonstrated in B lymphocytes (14, 15).

Thymocytes are endowed with several Ca2+-regulated signaling pathways, including Ca2+/calmodulin-dependent protein kinases (CaMKs)3 and the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which may contribute to discrimination between different Ca2+ signals. Calcineurin has been found important for positive selection (16, 17, 18, 19, 20) and in lowering the threshold for negative selection (10, 19). The role of CaMKs in T cell development is less clear. Of these, the CaMK type IV/Gr (CaMKIV/Gr) is of particular interest as an effector of Ca2+ signaling in thymocytes. CaMKIV/Gr expression is restricted to a few tissues, most notably T lymphocytes and neurons (21). In T lymphocytes, CaMKIV/Gr expression is developmentally regulated and is highest in CD4+CD8+ double-positive (DP) thymocytes (22, 23). CaMKIV/Gr is potently activated following TCR engagement (22, 24). It has been implicated in mediating Ca2+-dependent expression of genes encoding lymphokines, TNF family members, and immediate early activation products (25, 26, 27). In this study, we provide evidence for a role for CaMKIV/Gr in sensitizing thymocytes to selection events triggered by low-affinity peptide ligands.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CaMKIV/Gr-deficient mice were derived by targeted disruption of exon III of CaMKIV/Gr gene, as detailed elsewhere (28). Thymocytes and peripheral T cells of mutant mice lacked CaMKIV/Gr expression, as detected by immunoblotting with CaMKIV/Gr Abs. Mice were backcrossed for six to eight generations on C57BL/6(H2b) and B6.AKR(H2k) backgrounds. Wild-type (WT), heterozygote (Het), and knockout (KO) littermate mice were derived by mating of Het parents. All mouse protocols were in accordance with National Institutes of Health guidelines and approved by the Animal Care and Use Committee of Washington University School of Medicine (St. Louis, MO).

The 3.L2 TCR-transgenic mice (3.L2tg) express a receptor specific for the minor d allele of the {beta}-chain of murine hemoglobin, amino acids 64–76 (Hb{beta}d (64–76)), in the context of the MHC class II molecule I-Ek (29). The 3A9 TCR-transgenic mice express a receptor specific for a hen egg-white lysozyme (HEL) peptide, amino acids 46–61, presented by the MHC class II molecule I-Ak (30). The H-Y TCR-transgenic mice express a receptor that recognizes a male-specific Ag presented by the MHC class I molecule H-2b (31). Transgenic mice expressing a membrane form of HEL (mHEL) containing Hb{beta}d (64–76) as an epitope tag were derived as previously described (4). The mHEL transgene is controlled by the MHC-E{alpha} promoter, limiting expression to all class II-positive cells (4). Transgenic mice expressing the A72- and I72-altered peptide ligands of Hb{beta}d (64–76) were similarly derived (4). The relative expression level of transgenic mHEL proteins was equivalent, as determined by flow cytometric analysis and immunohistochemistry (4). The 3.L2tg, 3A9, and mHEL/Hb transgenic mice were bred to CaMKIV/Gr-deficient mice on B6.AKR background (F6 generation), while H-Y TCR-transgenic mice were bred to CaMKIV/Gr-deficient mice on a mixed 129/SvJ x C57BL/6 background. The progeny were genotyped by transgene-specific PCR analysis of purified tail digest DNA. H-Y transgenic mice were screened by flow cyotmetry using anti-H-Y clonotypic mAb (T3.70). TCR and mHEL/Hb transgenic mice used in this study were heterozygous for those transgenes. Mice were analyzed at ages 4–7 wk.

Antibodies

Fluorochrome-conjugated or biotinylated mAbs directed against the following murine Ags were obtained from BD PharMingen (San Diego, CA): TCR{beta}, CD3{epsilon}, CD4, CD5, CD8{alpha}, CD69, and B220. 3.L2, 3A9, and H-Y TCR clonotypic mAbs were generated and used as described (29, 31, 32). Secondary streptavidin-fluorochrome conjugates were from Caltag Laboratories (Burlingame, CA). Rabbit polyclonal anti-extracellular signal-related kinase (ERK)1,2 Abs and mouse anti-phospho-ERK mAb were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-phospho-CREB Abs were from Upstate Biotechnology (Lake Placid, NY) and rabbit polyclonal anti-CREB Abs were from Cell Signaling Technology (Beverly, MA).

Flow cytometry

Single cell suspensions of thymocytes or splenocytes were stained in FACS buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide) using the following protocol. Aliquots of cells (106/sample in 100 µl of FACS buffer) were placed in polypropylene culture tubes (12 x 75 mm) and incubated on ice for 1 h with the biotinylated or directly labeled Abs. For biotinylated Abs, cells were then washed once with 3 ml of FACS buffer and incubated for 30 min on ice with streptavidin-fluorochrome conjugates (Tricolor-streptavidin or PE-streptavidin; Caltag Laboratories), as appropriate. Cells were washed again, fixed for 18–24 h in FACS buffer plus 1% paraformaldehyde, and analyzed on a FACScan (BD Biosciences, Mountain View, CA) flow cytofluorometer using CellQuest (BD Biosciences) software. Samples were gated on live cells and 105 live cell events per sample were collected.

T cell proliferation assays

Cross-linking of mAb to tissue culture plates was achieved by first coating the plates overnight with polyclonal goat anti-hamster Abs (The Jackson Laboratory, Bar Harbor, ME) at 10 µg/ml in a carbonate coating buffer, pH 9.4. After washing, the plates were incubated with anti-TCR{beta} mAb for 90 min at the indicated concentrations. Splenocytes were suspended at 5 x 105 cells/well and incubated for 48 h with plate-bound anti-TCR{beta} mAb. The cells were then pulsed with 0.4 µCi/well [3H]TdR for 18 h and harvested. Proliferation was measured as cpm incorporated (mean of triplicate wells). Ag-specific proliferation was performed using total splenocytes incubated with increasing concentrations of Hb{beta}d (64–76) peptide.

Northern blot analysis

Total RNA was derived from freshly isolated thymocytes that were either left untreated or stimulated with ionomycin (1 µM) and PMA (20 ng/ml) for 1 or 3 h. RNA (10 µg/lane) was loaded on a formaldehyde gel, resolved by electrophoresis, and transferred to Hybond-XL membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were hybridized with the indicated probes overnight at 42°C in ULTRAhyb solution (Ambion, Austin, TX). The membranes were reprobed for GAPDH transcripts to confirm equal loading.

Immunoblotting

For evaluation of phospho-CREB and phospho-ERK induction following TCR signaling, thymocytes of WT, Het, and KO littermate mice were suspended at 107 cells/ml in RPMI 1640 medium at 37°C and stimulated for the indicated time periods with 10 µg/ml of an anti-CD3{epsilon} mAb (mAb 145-2C11; BD PharMingen) together with 10 µg/ml of a secondary cross-linking Ab. For immunblotting, whole cell lysates (107 cells/sample) were resolved by SDS-PAGE, then transferred to nitrocellulose and probed with one or more of the following Abs, as indicated: mouse anti-CaMKIV/Gr catalytic domain mAb, rabbit polyclonal anti-ERK and anti-phospho-ERK Abs, and anti-CREB and phospho-CREB Abs. The blots were developed using HRP-conjugated secondary Abs and enzyme-linked chemiluminescence (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Impaired thymocyte maturation in CaMKIV/Gr-deficient mice

The role of CaMKIV/Gr in T cell development was addressed using CaMKIV/Gr-deficient mice derived by targeted gene inactivation (28). Homozygous-deficient (KO) mice lacked CaMKIV/Gr expression in thymocytes and in peripheral T cells, while Het mice expressed CaMKIV/Gr at 50% of WT levels (Fig. 1Goa). Thymic cellularity of CaMKIV/Gr KO mice was not statistically different from that of WT littermate controls. However, flow cytometric analysis revealed a reduction in the percentages of CD4 and CD8 single-positive (SP) thymocytes in CaMKIV/Gr KO mice when compared with WT littermates (mean reduction 34% and 35%, respectively; n = 12 pairs of WT and KO mice, p < 0.001) (Fig. 1Gob). There was a concomitant increase in the CD4+CD8+ (DP) compartment. Examination of TCR expression on CD4-SP thymocytes revealed decreased numbers of cells expressing high levels of TCR in KO mice compared with WT littermates. However, the level of TCR expression on KO SP cells was not affected (Fig. 1Gob). We also analyzed the expression in KO thymocytes of developmentally regulated Ags such as CD5 and CD69, whose levels are up-regulated in the course of positive selection. The percentage of CD5+ and CD69+ cells was decreased in KO mice to an extent commensurate with the decrease in SP thymocytes. However, the level of expression of both markers was not affected (Fig. 1Goc). These results are consistent with decreased production in CaMKIV/Gr KO mice of SP thymocytes.



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  		FIGURE 1. Analysis of thymocyte development in CaMKIV/Gr-deficient mice. a, Immunblot analysis of CaMKIV/Gr expression in WT, Het, and CaMKIV/Gr KO thymocytes using anti-CaMKIV/Gr catalytic domain mAb. The {alpha} and {beta} isoforms of CaMKIV/Gr are indicated by arrowheads. b, Dot plots from flow cytometric analysis of thymocytes of CaMKIV/Gr WT and KO mice stained with anti-CD4-PE and CD8-FITC mAbs. The numbers indicate percentage of cells in the respective quadrant. Shown below are histograms of TCR expression on CD4-SP WT and KO thymocytes (R1 region in dot plots). TCR expression was analyzed using anti-TCR{beta}-chain mAb. Peak fluorescence intensities of CD4, CD8, and TCR were similar in WT and CaMKIV/Gr KO thymocytes. Results are representative of 12 pairs of WT and CaMKIV/Gr KO mice. c, Expression of CD5 and CD69 in WT and KO thymocytes. Histogram analysis of CD5 and CD69 expression on total thymocyte population of WT and CaMKIV/Gr KO thymocytes. Peak fluorescence intensities of CD5 and CD69 were similar in WT and KO mice.

 
Examination of the peripheral T cell compartment revealed a modest increase in spleen size of CaMKIV/Gr KO mice relative to WT controls. The percentages of CD4+ and CD8+ splenic T cells were mildly decreased in KO mice relative to WT controls (Fig. 2Goa). However, when corrected for splenic cellularity, the absolute numbers of CD4+ and CD8+ T cells were maintained. In contrast, the B cells were increased in KO mice. Both WT and KO splenocytes proliferated equally well upon stimulation with cross-linked anti-TCR mAb (Fig. 2Gob).



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  		FIGURE 2. Analysis of peripheral lymphocytes in CaMKIV/Gr-deficient mice. a, Dot plot analysis of splenic cells of WT and CaMKIV/Gr KO mice stained with anti-CD4-PE and anti-CD8-FITC mAbs. Shown below are histograms of TCR expression on CD4-SP splenocytes in R1 in dot plots, as revealed by staining with anti-TCR mAb. Results are representative of 20 pairs of WT and CaMKIV/Gr KO mice. b, Proliferation of total splenic T cells of WT and KO mice to cross-linked anti-TCR{beta} mAb. Results represent mean values ± SEM of proliferative responses obtained in six pairs of WT and littermate CaMKIV/Gr KO mice.

 
Positive and negative selection in TCR-transgenic x CaMKIV/Gr KO mice

The influence of CaMKIV/Gr deficiency on T cell selection events was further examined using transgenic mice expressing TCR with defined specificity. The 3.L2tg mice express T cells specific for the minor d allele of the {beta}-chain of murine hemoglobin, amino acids 64–76, in the context of the MHC class II molecule I-Ek (29). Positive selection of 3.L2tg+ thymocytes on endogenous peptides plus I-Ek results in mature CD4+ T cells that express high levels of the transgenic TCR. Analysis of 3.L2tg x CaMKIV/Gr KO mice revealed that while the thymus size was not significantly different from WT controls, the percentage of CD4-SP thymocytes was decreased by 45% in KO mice (n = 10 pairs of WT and KO mice, p < 0.001) (Fig. 3Goa). There was a corresponding increase in the CD4+CD8+ (DP) compartment. Staining with a 3.L2 TCR clonotypic mAb showed marked reduction (57%) in the generation of 3.L2tg TCRhigh CD4-SP thymocytes in 3.L2tg x CaMKIV/Gr KO mice, consistent with the decreased generation of CD4-SP thymocytes.



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  		FIGURE 3. Impaired thymocyte-positive selection in CaMKIV/Gr-deficient, MHC class II-restricted TCR-transgenic mouse models. a, Flow cytometric analysis of thymocytes of CaMKIV/Gr WT and KO mice expressing the 3.L2tg TCR. A dot plot of CD4 vs CD8 expression in thymocytes of representative WT and KO 3.L2 TCR-transgenic mice is shown. From these plots, the CD4-SP regions (R1) were selected for further analysis of 3.L2 TCR expression using biotinylated 3.L2 clonotypic mAb and Tricolor streptavidin, which is shown in the histograms below. The percentages reported in the single histograms of 3.L2tg TCR (and all subsequent histograms of other transgenic TCR) are in reference to total thymocytes. Results are representative of 10 pairs of 3.L2tg WT and CaMKIV/Gr KO mice. b, Flow cytometric analysis of thymocytes of WT and KO 3A9 TCR-transgenic mice, which were analyzed similar to A using 3A9 TCR clonotypic mAb. Results are representative of five pairs of 3A9 WT and CaMKIV/Gr KO mice.

 
The impact of CaMKIV/Gr deficiency on positive selection was further examined using the 3A9 TCR-transgenic mice, which express T cells specific for HEL (46–61) in the context of I-Ak (30). Fig. 3Gob demonstrates that the percentage of CD4-SP thymocytes was decreased by about one-third in 3A9 x CaMKIV/Gr KO mice relative to 3A9 WT controls. Staining with a 3A9 TCR clonotypic mAb revealed a corresponding reduction in the generation of 3A9 TCRhigh CD4-SP thymocytes in 3A9 x CaMKIV/Gr KO mice. Together, the results obtained in the 3.L2tg and 3A9 TCR-transgenic mice confirmed that CaMKIV/Gr deficiency impairs positive selection of MHC class II-restricted, TCR-transgenic thymocytes.

The impact of CaMKIV/Gr deficiency on MHC class I-restricted positive selection was examined using TCR-transgenic mice expressing the H-Y TCR, which is specific for the H-Y male Ag in the context of H-2b (31). Thymocytes expressing the H-Y-specific TCR are positively selected along the CD8 lineage in female mice, but are deleted in male mice. Analysis of H-Y TCR+ female transgenic mice demonstrated that the percentage of CD8-SP thymocytes was decreased in CaMKIV/Gr KO mice by ~50% as compared with WT mice (Fig. 4Go). Staining with a H-Y TCR clonotypic mAb also showed a corresponding reduction in the generation of H-Y TCRhigh CD8-SP thymocytes in H-Y TCR-transgenic x CaMKIV/Gr KO, consistent with the decreased generation of CD8-SP thymocytes (Fig. 4Go). The percentage of CD4-SP cells was also decreased in H-Y TCR+ KO mice, while the percentage of DP cell thymocytes was increased. These results are consistent with a block in DP to SP transition in CaMKIV/Gr-deficient H-Y TCR-transgenic mice that impairs both MHC class I- and class II-restricted positive selection events.



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  		FIGURE 4. Impaired thymocyte-positive selection in a CaMKIV/Gr-deficient, MHC class I-restricted TCR-transgenic mouse model. Dot plots from flow cytometric analysis of thymocytes of WT and KO H-Y TCR-transgenic female mice, stained for CD4 and CD8, showing a reduction in both CD8 and CD4-SP thymocyte compartment. From these plots, the CD8-SP regions (R1) were selected for further analysis of H-Y TCR expression by staining with clonotypic H-Y TCR mAb, shown in the histograms below. Results are representative of five WT and three CaMKIV/Gr KO H-Y TCR-transgenic female mice.

 
To examine the consequence of CaMKIV/Gr deficiency on negative selection, 3.L2tg x CaMKIV/Gr KO mice were crossed with transgenic mice expressing mHEL into which Hb{beta}d (64–76) had been engineered (4). The mHEL/Hb{beta}d (64–76) transgene is under control of E{alpha} promoter, directing expression into APCs, including those in the thymus. The mHEL/Hb{beta}d (64–76) protein is well expressed and efficiently processed, enabling presentation of Hb{beta}d (64–76) in the context of I-Ek. When 3.L2tg mice are crossed with those expressing mHEL/Hb{beta}d (64–76), 3.L2 TCR+ thymocytes are completely deleted (4). Fig. 5Goa demonstrates that 3.L2 TCR+ thymocytes of CaMKIV/Gr WT and KO mice were deleted to a similar extent when exposed to mHEL//Hb{beta}d (64–76), indicating that clonal deletion of 3.L2tg thymocytes by Hb{beta}d (64–76) was not impaired by CaMKIV/Gr deficiency.



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  		FIGURE 5. CaMKIV/Gr deficiency does not impair Ag-specific negative selection. a, Negative selection in 3.L2tg mice. Dot plot analysis of CD4 and CD8 staining on thymocytes of 3.L2tg x mHEL/Hb{beta}d (64–76) and 3.L2tg x mHEL/Hb{beta}d (64–76) x CaMKIV/Gr KO mice. From these plots, the CD4-SP regions (R1) were selected for further analysis of 3.L2 clonotypic TCR expression. The histogram on the left shows overlay of 3.L2tg x mHEL/Hb{beta}d (64–76) (solid line) and 3.L2tg controls (dotted line). Histogram on the right shows overlay of 3.L2tg x mHEL/Hb{beta}d (64–76) x CaMKIV/Gr KO (solid line) and 3.L2tg x CaMKIV/Gr KO (dotted line). Results are representative of three pairs of mice. b, Negative selection in male H-Y TCR-transgenic mice. Dot plot analysis of CD4 and CD8 expression on thymocytes of male H-Y TCR-transgenic and H-Y TCR-transgenic x CaMKIV/Gr KO mice. Left histogram shows overlay of H-Y TCR staining in CD8-SP thymocytes of an H-Y TCR-transgenic male (solid line) and a female control (dotted line). Right histogram shows the equivalent staining in an H-Y TCR-transgenic x CaMKIV/Gr KO male (solid line) and an H-Y TCR-transgenic x CaMKIV/Gr KO female control (dotted line). Results are representative of four pairs of mice.

 
The influence of CaMKIV/Gr deficiency on negative selection was further examined using male H-Y TCR-transgenic mice. In male mice, engagement of the H-Y TCR by the H-Y Ag results in deletion of H-Y TCR+ thymocytes, leading to a drastic reduction in the number of DP and SP thymocytes (31). Examination of thymi of H-Y TCR+ WT and CaMKIV/Gr KO male mice revealed equally reduced cellularity and thymocyte deletion (Fig. 5Gob). This indicated that CaMKIV/Gr deficiency did not affect negative selection of H-Y TCR+ thymocytes by the H-Y Ag.

Altered selection threshold in CaMKIV/Gr KO mice

Because Hb{beta}d (64–76) and the H-Y Ag are strong deleting ligands, we sought to examine the effect of CaMKIV/Gr on clonal deletion by weaker ligands. To that end, we employed altered peptide ligands derived from Hb{beta}d (64–76) in which the asparagine residue at position 72 of Hb{beta}d (64–76) (N72) has been changed to either isoleucine (I72) or alanine (A72). Both I72 and A72 are antagonists whose relative affinity for the 3.L2 TCR follows the order N72(WT) > I72 > A72 (4). The I72 peptide induces strong negative selection of 3.L2tg thymocytes, whereas the A72 peptide induces weak to moderate negative selection.

Both I72 and A72 peptides were engineered into mHEL proteins and expressed in APCs of transgenic animals under control of I-E{alpha} promoter (4). Expression levels of mHEL/I72 and mHEL/A72 were equivalent to those achieved with mHEL/Hb{beta}d (64–76) (Ref. 4 and data not shown). Transgenic mice expressing mHEL/I72 (I72tg) and mHEL/A72 (A72tg) were crossed with 3.L2tg x CaMKIV/Gr WT and KO mice, and the progeny was examined for deletion of 3.L2tg thymocytes. Studies on 3.L2tg x I72tg and 3.L2tg x I72tg x CaMKIV/Gr KO mice demonstrated that the I72 peptide induced virtually complete deletion of 3.L2 TCR+CD4-SP thymocytes in both WT and KO mice, indicating that CaMKIV/Gr deficiency did not impair deletion by I72 (data not shown). The A72 peptide induced moderate deletion of 3.L2 TCR+CD4-SP thymocytes in CaMKIV/Gr WT mice. While 3.L2 TCR+CD4-SP thymocytes are decreased in CaMKIV/Gr KO mice due to impaired positive selection, exposure to A72 would be expected to induce a further decrease in their number. Rather, both the CD4-SP and the 3.L2 TCR+CD4-SP thymocytes were increased in 3.L2tg x A72tg x CaMKIV/Gr KO relative to 3.L2tg x CaMKIV/Gr KO mice (CD4-SP, 8.20 ± 0.7% vs 5.9 ± 0.3%, p = 0.003; 3.L2 TCR+CD4-SP, 4.3 ± 0.6 vs 2.8 ± 0.2, p = 0.01, n = 7 and 10, respectively) (Fig. 6Go, a and b). Consistent with these findings, analysis of splenocytes of 3.L2tg x A72tg x CaMKIV/Gr revealed marked increase of 3.L2 TCR+CD4+ splenocytes in 3.L2tg x CaMKIV/Gr KO x A72tg relative to 3.L2tg x CaMKIV/Gr KO mice (5.5 ± 0.4 vs 2.5 ± 0.4, p < 0.001, n = 4 and 6, respectively) (Fig. 6Goc). These results indicated that the A72 peptide, which normally induces weak to moderate negative selection, promoted positive selection in the context of CaMKIV/Gr deficiency.



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  		FIGURE 6. Altered selection threshold in CaMKIV/Gr KO mice. a, Negative selection in 3.L2tg x A72tg mice. Dot plot analysis of CD4 and CD8 staining on thymocytes of 3.L2tg x A72tg and 3.L2tg x A72tg x CaMKIV/Gr KO mice. The histogram on the left shows overlay of 3.L2tg x A72tg (solid line) and 3.L2tg controls (dotted line). Histogram on the right shows overlay of 3.L2tg x A72tg x CaMKIV/Gr KO (solid line) and 3.L2tg x CaMKIV/Gr KO (dotted line). b and c, Frequency of 3.L2 TCR+CD4-SP thymocytes (b) and splenocytes (c) in WT and KO strains expressing either the 3.L2tg or 3.L2tg x A72tg. d, Proliferative responses of splenocytes of the respective strains to Hb{beta}d (64–76). Results were normalized for the number of 3.L2 TCR+ splenocytes present in culture and expressed as cpm/103 3.L2 TCR+ splenocytes.

 
To determine whether the increased positive selection in 3.L2tg x A72tg x CaMKIV/Gr KO mice results in the production of functional 3.L2 TCR+CD4+ splenocytes, we compared the proliferative responses to Hb{beta}d (64–76) peptide of splenocytes of 3.L2tg, 3.L2tg x CaMKIV/Gr KO, 3.L2tg x A72tg, and 3.L2 x A72tg x CaMKIV/Gr KO. The results, normalized for 3.L2 TCR+CD4+ cell count and shown in Fig. 6God, revealed that splenocytes of 3.L2tg and 3.L2tg x CaMKIV/Gr KO mice proliferated equally well in response to Hb{beta}d (64–76) peptide. Importantly, splenocytes of 3.L2tg x A72tg and 3.L2tg x A72tg x CaMKIV/Gr KO mice also proliferated equally well in response to Hb{beta}d (64–76) peptide, indicating that they were functionally equivalent. The presence of the A72tg was associated with a modest shift to the right in the peptide concentration/proliferative response curves of both WT and KO splenocytes that was of similar magnitude (Fig. 6God). This is consistent with the previously reported peripheral antagonism by the A72 peptide of proliferative responses induced by the native Hb{beta}{delta} (64–76) peptide (29, 31, 32). Overall, these results confirm that the promotion of positive selection by the A72tg in 3.L2 x CaMKIV/Gr KO results in the production of functional 3.L2 TCR+ T cells.

Defective induction of Ca2+-regulated genes in CaMKIV/Gr KO thymocytes

To elucidate mechanisms by which CaMKIV/Gr regulates thymocyte selection, we examined Ca2+-regulated gene expression in thymocytes of CaMKIV/Gr KO mice. Previously, studies using T cell lines have demonstrated that CaMKIV/Gr mediates Ca2+-dependent transcriptional activation of several genes relevant to thymocyte selection. These encode the TNF family members CD40 ligand (CD40L) and TNF-{alpha} and the orphan steroid receptor Nur77, all of which have been implicated in negative selection (33, 34, 35). Fig. 7Goa demonstrates that, compared with WT thymocytes, CaMKIV/Gr KO thymocytes expressed markedly lower levels of CD40L, TNF-{alpha}, and Nur77 transcripts following stimulation with phorbol ester and Ca2+ ionophore. Transcripts of other Ca2+-regulated genes such as c-fos were modestly decreased, while those of c-jun were spared. These results revealed a selective impairment of Ca2+-regulated gene transcription in CaMKIV/Gr KO thymocytes.



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  		FIGURE 7. Impaired Ca2+-regulated gene expression in CaMKIV/Gr KO thymocytes. a, Northern blot analysis of total RNA isolated from untreated and phorbol ester and calcium ionophore-treated thymocytes of WT, Het, and CaMKIV/Gr KO mice. Results are representative of three separate experiments. b, Normal MAP kinase activation and CREB phosphorylation in CaMKIV/Gr KO thymocytes. Thymocytes of WT and KO mice were stimulated with cross-linked anti-CD3{epsilon} mAb and then examined for ERK and CREB phosphorylation by immunoblotting with specific phosphoantibodies. The total amount of ERK and CREB proteins was subsequently determined by immunoblotting with anti-ERK and anti-CREB Abs. Results are representative of three separate experiments.

 
The effects of CaMKIV/Gr on thymocyte selection overlap with those of previously reported lesions in other signaling pathways. In particular, ERK1 and ERK2 have previously been demonstrated to promote positive selection (36, 37, 38). To determine whether CaMKIV/Gr deficiency impairs ERK activity, WT and KO thymocytes were stimulated with anti-CD3{epsilon} mAb and then examined for ERK1 and ERK2 activation, as evidenced by activation loop-specific phosphorylation of ERK1 and ERK2. Results revealed that ERK activation proceeded normally in CaMKIV/Gr KO thymocytes (Fig. 7Gob). This indicated that the impact of CaMKIV/Gr deficiency on thymocyte selection was unlikely to be due to impaired ERK activity.

CaMKIV/Gr is also known to be a prominent activator of CREB, which it phosphorylates on the regulatory serine 133 residue (39, 40). Its deficiency is associated with impaired Ca2+-dependent CREB phosphorylation in neurons (28, 41). Because CREB has been implicated in regulating thymocyte development (42, 43), we examined whether the effects of CaMKIV/Gr deficiency on thymocyte development correlate with impaired CREB activation. Thymocytes of WT and KO mice were stimulated with anti-CD3{epsilon} mAb and then examined for CREB activation, as evidenced by its phosphorylation on serine 133. Results (Fig. 7Gob) revealed that CREB phosphorylation proceeded normally in CaMKIV/Gr KO thymocytes, indicating that unlike the case of neurons, CREB activation in CaMKIV/Gr KO thymocytes is rescued by other CREB-activating kinase(s).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we provide evidence for an important role for CaMKIV/Gr in thymocyte selection. First, CaMKIV/Gr deficiency was associated with impaired generation of both CD4-SP and CD8-SP thymocytes. The defect involved a block in the transition from DP into SP thymocytes, as evidenced by a corresponding increase in the number of DP thymocytes. This block did not involve lineage commitment per se, but rather trophic signal(s) that promotes maturation of both lineages. Significantly, CaMKIV/Gr KO mice bred onto MHC class I (H-Y)- and class II (3.L2 and 3A9)-restricted TCR-transgenic backgrounds exhibited a similar deficit in the generation of TCR-transgenic SP thymocytes. These results are all consistent with impaired thymocyte-positive selection in CaMKIV/Gr KO mice.

CaMKIV/Gr deficiency did not affect negative selection by high-affinity peptide ligands. This was evidenced by the effective deletion of CaMKIV/Gr KO thymocytes bearing either MHC class I (H-Y)- or II (3.L2)-restricted transgenic TCR upon exposure to their specific Ags. However, by using altered peptide ligands that vary in their affinity for the 3.L2 TCR, it could be demonstrated that CaMKIV/Gr deficiency altered the outcome of negative selection by a low-affinity ligand. This was evidenced by the abrogation in 3.L2tg x CaMKIV/Gr KO mice of weak negative selection by the A72 peptide, which instead acted to promote positive selection. Because positive selection is normally an attribute of peptides of lower affinity than A72 for the 3.L2 TCR, this indicated that CaMKIV/Gr deficiency rendered the A72 peptide functionally equivalent to a peptide of lower affinity for the 3.L2 TCR.

The quantitative/avidity model of thymocyte selection predicts that a majority of peptides encountered during development have either no affinity (null) or low affinity (positively selecting) for a given TCR, while a small population of peptides exhibits high enough affinity to initiate negative selection (3, 44). Given that CaMKIV/Gr KO mice suffer impairment of selection processes typically driven by lower-affinity peptide/TCR interactions including positive selection and weak negative selection, it can be concluded that CaMKIV/Gr-regulated pathways function to sensitize thymocytes to selection by lower-affinity peptides. This may be due to a requisite role for CaMKIV/Gr in a subset of signaling events triggered by low-affinity peptides. Accordingly, CaMKIV/Gr deficiency raises the threshold for selection such that a large number of low-affinity peptides that normally induce positive selection are rendered functionally null, leading to impaired positive selection. Of the smaller group of peptides that mediate negative selection, the effectiveness of those at the lower end of the affinity spectrum is attenuated, leading to impaired weak negative selection. Peptides at the threshold of negative selection such as A72 may instead promote positive selection in the context of CaMKIV/Gr deficiency. However, this is insufficient to rescue the defect in positive selection possibly due to the low frequency of such peptides. Only negative selection by a minority of peptides at the high end of the affinity spectrum, such as the H-Y Ag and Hb{beta}d (64–76), is spared.

The expression profile and activation mechanism of CaMKIV/Gr make it particularly suited for promotion of thymocyte selection by low-affinity peptides. CaMKIV/Gr levels are highest in DP thymocytes, which are the target of thymocyte selection events. CaMKIV/Gr is itself a component of an independent CaMK cascade that includes the upstream activators CaMKK{alpha} and CaMKK{beta} (45). Signal amplification by this cascade may allow CaMKIV/Gr to be activated at lower Ca2+ concentrations, such as those triggered by low-affinity ligands. CaMKIV/Gr deficiency did not impair transient, high-intensity activation of other kinase cascades involved in thymocyte selection, such as those of the mitogen-activated protein (MAP) kinases. A more subtle crosstalk between the CaMK and MAP kinase cascade cannot however be ruled out and requires further investigation.

The thymic phenotype observed in CaMKIV/Gr KO mice contrasts with that previously reported for mice expressing a dominant-negative (DN) CaMKIV/Gr mutant in thymocytes (46). Thymi of the latter mice were profoundly hypocellular, and their T cells proliferated poorly upon stimulation with anti-CD3 mAbs. The discrepant phenotype between the CaMKIV/Gr KO and DN CaMKIV/Gr mutant mice may be due to inhibitory interference by the DN CaMKIV/Gr mutant with other protein kinases that ameliorate the impact of CaMKIV/Gr deficiency on thymic development. These are most likely to be other components of the CaMK cascade itself. Both CaMKK{alpha} and CaMKK{beta} physically interact with CaMKIV/Gr by means of a specialized domain (47). Hence, overexpression of the DN CaMKIV/Gr mutant is likely to tie up both upstream activating kinases in complexes with the inactive mutant, leading to inhibition of their CaMKIV/Gr-dependent and independent functions. The global role of the CaMK cascade in thymic selection is currently being addressed using mutant mice lacking different component kinases.

A principal function of CaMKIV/Gr is Ca2+-dependent transcriptional activation (45). Consistent with this function is the decreased transcription in CaMKIV/Gr KO thymocytes of several Ca2+-regulated genes. Some affected genes such as CD40L, TNF, and Nur77 have been previously implicated in negative selection (33, 34, 35), suggesting that their defective transcription may contribute to the impaired weak negative selection in CaMKIV/Gr KO mice. Altered transcription of other currently unknown genes may similarly contribute to impaired positive selection in CaMKIV/Gr KO mice.

CaMKIV/Gr regulates several transcriptional activators, coactivators, and corepressors, including CREB (39, 40), p300/CREB-binding protein (48), myocyte enhancer factor 2 (MEF2) (27), and histone deacetylases (49). CaMKIV/Gr deficiency appeared to selectively impact some factors but not others. TCR-triggered CREB phosphorylation proceeded normally in CaMKIV/Gr-deficient thymocytes, suggesting that this pathway was rescued by the action of other CREB kinases. In contrast, Ca2+ activation of MEF2 factors appeared impaired, as evidenced by decreased transcription of Nur77, a prototypic, MEF2-regulated immediate-early activation gene (27, 50). Because evaluation of Ca2+-dependent gene transcription was performed under conditions approximating strong TCR/CD3 signaling induced by high-affinity ligands, a more dramatic impact of CaMKIV/Gr deficiency on select Ca2+-regulated transcriptional events may be revealed by using low-affinity ligands or surrogate stimulation paradigms. Such studies, currently ongoing, may also be informative into the role of individual Ca2+-regulated factors in mediating the effects of CaMKIV/Gr on thymocyte selection.

Finally, the interplay between the CaMK cascade and other Ca2+ signaling pathways in directing the selection outcome in thymocytes merits attention. Some pathways, such as the CaMK cascade (this study) and the calcineurin/NF-AT module (16, 17, 18, 19, 20), appear to be preferentially employed by thymocytes to decode Ca2+ signals associated with positive and weak negative selection. Others, such as the NH2-terminal Jun kinase, may be recruited by stronger Ca2+ responses associated with negatively selecting ligands (51). It will be important to elucidate the different affinity thresholds at which these pathways are recruited and how different pathways interact to achieve a particular selection outcome.


   Acknowledgments
 
We thank Andrew Chan and Emil Unanue for critical review of the manuscript. We thank Paul Allen for the gift of 3.L2, 3A9, and mHEL transgenic mice and for 3.L2 clonotypic mAb; Andrew Chan for the gift of H-Y transgenic mice; H. S. Teh for the H-Y TCR clonotypic mAb; Emil Unanue for the 3A9 clonotypic mAb; and Kenneth Murphy for support with flow cytometry.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants 5RO1HD35694 (to T.A.C.) and RO1AI47154 (to C.B.W.), and by March of Dimes Grant 5-722 (to C.B.W.). Back

2 Address correspondence and reprint requests to Dr. Talal Chatila, Departments of Pediatrics and Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8208, St. Louis, MO 63110. E-mail address: chatila{at}kids.wustl.edu Back

3 Abbreviations used in this paper: CaMK, Ca2+/calmodulin-dependent protein kinase; CaMKIV/Gr, CaMK type IV/Gr; CD40L, CD40 ligand; DN, dominant-negative; DP, double positive; ERK, extracellular signal-related kinase; HEL, hen egg-white lysozyme; Het, heterozygote; KO, knockout; MAP, mitogen-activated protein; MEF2, myocyte enhancer factor 2; mHEL, membrane form of HEL; SP, single positive; WT, wild type. Back

Received for publication August 15, 2001. Accepted for publication October 1, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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