Function of CD3{epsilon}-mediated Signals in T Cell Development

Connie L. Sommersa, Jan B. Dejarnettea, Kun Huanga, Jan Leeb, Dalal El-Khourya, Elizabeth W. Shoresb, and Paul E. Lovea
a Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
b Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

Correspondence to: Paul E. Love, Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. Tel:301-402-4946 Fax:301-480-6302


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

The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3{gamma}, CD3{delta}, CD3{epsilon}, and {zeta}) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3{epsilon} ITAM-mediated signals in T cell development by genetically reconstituting CD3{epsilon}-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3{epsilon} are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for {zeta} chain. Unexpectedly, analysis of TCR-transgenic/CD3{epsilon}-mutant mice reveals a potential role for CD3{epsilon} signals in T cell survival.

Key Words: TCR, CD3epsilon, development, mice, cell survival


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

Signals transduced by the TCR initiate a broad range of mature T cell responses. Signaling by the TCR and its precursor, the pre-TCR, are also required for thymocyte development and selection (1) (2) (3). The pre-TCR and TCR activate intracellular signaling cascades through semiconserved sequences of amino acids referred to as immunoreceptor tyrosine-based activation motifs (ITAMs). After receptor engagement, phosphorylation of TCR-ITAM tyrosines leads to the recruitment and activation of src homology 2 (SH2) domain–containing kinases (2). The pre-TCR and TCR contain four distinct signal-transducing subunits—CD3{gamma}, CD3{delta}, CD3{epsilon}, and {zeta}—assembled as dimers: {gamma}/{epsilon}, {delta}/{epsilon}, and {zeta}{zeta}. Each of the CD3 subunits contains a single ITAM, whereas {zeta} chain contains three ITAMs.

Analysis of the function of TCR-ITAMs has revealed that individual motifs bind with different affinities to the same effector (ZAP-70) and bind selectively to other potential effector molecules (4) (5). On the other hand, isolated ITAMs have been shown to be independently capable of initiating a broad range of T cell responses, including proliferation and cytokine synthesis (6) (7).

Experimental data support the idea that at least some TCR-ITAMs are functionally equivalent with respect to the signaling requirements for T cell development. For example, mature T cells are still generated in mice that express TCRs lacking the three {zeta} chain ITAMs, and these cells appear functionally competent (8) (9) (10). In contrast to {zeta} chain, the importance of signals transduced by the CD3 subunits has not been as rigorously addressed. Chimeric (Tac/{zeta} or Tac/{epsilon}) transgenes were found to be capable of independently mimicking pre-TCR signals and some TCR signals; however, it is unknown if endogenous {zeta} and CD3{epsilon} chains contributed to these results (11). Mice lacking CD3{gamma}, CD3{delta}, or CD3{epsilon} exhibit various degrees of developmental arrest, demonstrating a clear role for these proteins in T cell maturation (12) (13) (14) (15). However, the importance of the CD3 chains to the TCR signaling response is difficult to assess in knockout mice, as these proteins are also required for TCR assembly and surface expression (12) (13) (14) (15).

Among the CD3 subunits, CD3{epsilon} is the most likely candidate for performing a critical role in the TCR signaling response. CD3{epsilon} is represented twice in the TCR complex, serving as a component of both the {gamma}/{epsilon} and {delta}/{epsilon} dimers. In addition, after TCR cross-linking, CD3{epsilon} and {zeta} are the predominant tyrosine-phosphorylated TCR subunits. In this study, we assessed the importance of CD3{epsilon} signals during T cell development by genetically reconstituting CD3{epsilon}-deficient mice with transgenes encoding either the wild-type (WT) or a mutant (signaling defective) form of the protein. The results demonstrate that signals transduced by CD3{epsilon} are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling. Interestingly, the phenotype of TCR-transgenic/CD3{epsilon}-mutant mice suggests a potential role for CD3{epsilon} signals in particular or TCR signals in general for the generation and/or survival of mature T cells.


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

Generation of CD3{epsilon}{Delta}/{Delta}, CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg, and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg Mice.
The generation of mice lacking expression of CD3{epsilon} (CD3{epsilon}{Delta}/{Delta}) and huCD2-CD3{epsilon} transgenic (tg) mice has been described previously (15). The huCD2-CD3{epsilon}M transgene was generated by mutating the murine CD3{epsilon} ITAM sequence in vitro using a synthetic oligonucleotide that substitutes phenylalanine (AAA) for tyrosine (ATA) at position 181. Transgenic founder lines that expressed levels of protein that most closely matched that of endogenous CD3{epsilon} were used in the experiments described here. TCR-transgenic mice used in these studies included the MHC class I–restricted TCRs H-Y (16) and P14 (17), which were maintained in the H-2Db background.

Western Blot Analysis.
Thymocytes were enumerated, washed twice in PBS, and resuspended in PBS at a concentration of 108 per milliliter. Thymocyte stimulations, immunoprecipitations, Western blotting, and polyacrylamide electrophoresis were performed as described (18) (19). Separated proteins were transferred to polyvinylidene difluoride membranes and blotted with antiphosphotyrosine mAb (4G10; Upstate Biotechnology) or monoclonal anti-CD3{epsilon} (HMT3.1) followed by anti–mouse IgG–horseradish peroxidase (Transduction Laboratories) or protein A–horseradish peroxidase (Transduction Laboratories), respectively, and detected by chemiluminescence (ECL; Amersham Pharmacia Biotech).

Multicolor Flow Cytometry and Measurement of Calcium Flux.
Single-cell suspensions of thymocytes or lymph node cells were processed, stained, and analyzed as described previously (8). Calcium flux measurements were performed as described (19).

Proliferation and Cytokine Assays.
Single-cell suspensions were prepared from thymi or lymph nodes in RPMI plus 10% FCS. CD4+CD8- T cells were enriched by panning after incubating cells with antibodies to CD8, B220, and MAC-1 for 30 min. Alternatively, CD8+CD4- thymocytes were enriched by panning after incubating cells with antibodies to CD4, B220, and MAC-1 for 30 min. Cells were washed, resuspended in RPMI plus 10% FCS, and placed on rabbit anti–mouse Ig–coated plates at 25°C, 5% CO2. Nonadherent cells were collected after 1–2 h. In some experiments, purified cells were labeled with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) before stimulation. Cells were washed twice in HBSS, resuspended at 4 x 106 cells/ml in HBSS and 50 ng/ml CFSE, incubated for 15 min at 37°C, and then washed twice in HBSS and resuspended in RPMI plus 10% FCS. Accessory cells and APCs were prepared from spleen suspensions of C57BL/6 mice. APCs were depleted of T cells with anti-Thy1.2 + C' and irradiated with 3,000 rads. For antibody-mediated stimulation, wells of 96-well flat-bottomed plates were coated with antibodies at the indicated concentrations (see Fig 3) overnight at 37°C and washed three times with sterile media. 105 responder T cells were added to the wells, incubated at 37°C for 40 h, and then pulsed with 1 µCi of [3H]thymidine for 8–12 h. For stimulation with peptide, 105 responder T cells were combined with 5 x 105 APCs (obtained from female mice) in flat-bottomed 96-well plates in the presence or absence of the peptide. Smcy peptide (KCSRNRQYL) was synthesized in the FDA Core Facility and was added to culture at the indicated concentrations (see Fig 3). Proliferation and cytokine assays were performed as described (8) (19). For IL-2 production, cells were stimulated as described above and culture supernatants were harvested after 48 h. IL-2 ELISAs were performed using the reagents and protocol obtained from PharMingen.



View larger version (34K):
[in this window]
[in a new window]
 
Figure 1. Phenotype of CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. Comparison of thymocytes (A) and lymph node cells (B) from control ({epsilon}+/+), {epsilon}{Delta}/{Delta}, {epsilon}{Delta}/{Delta}; {epsilon}-tg, and {epsilon}{Delta}/{Delta}; {epsilon}M-tg mice by FACS® analysis. Two-color plots show CD4/CD8 staining profiles. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are representative of three separate experiments. (C) Analysis of CD4-CD8- thymocyte subsets in CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. Left panels, two-color (CD25 vs. CD44) staining on gated (CD4-CD8-CD3-) thymocytes. Right panels, CD3 vs. TCR-{delta} staining on gated (CD4-CD8-) thymocytes.




View larger version (116K):
[in this window]
[in a new window]
 
Figure 2. (A) Analysis of CD3{epsilon} and CD3{epsilon}M protein tyrosine phosphorylation after stimulation of thymocytes with pervanadate. Thymocytes (108) from nontransgenic B6 mice (N.Thy), CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg ({epsilon}-tg), and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg ({epsilon}M-tg) mice were incubated for 5 min in medium lacking (-) or containing (+) pervanadate. Cell lysates were immunoprecipitated with anti-CD3{epsilon} (mAb 145-2C11). Immunoprecipitated proteins were resolved by 12% SDS-PAGE, followed by antiphosphotyrosine (mAb 4G10) Western blotting (top blot). Blots were subsequently stripped and reprobed with anti-CD3{epsilon} (mAb HMT3.1; bottom blot). (B) Genetic reconstitution of mice deficient for {zeta} and CD3{epsilon} ({zeta}-/- x {epsilon}{Delta}/{Delta}) with transgenes encoding WT or mutant CD3{epsilon} proteins. Two-color plots show CD4/CD8 staining profiles on total thymocytes or total lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show CD3 surface staining on total thymocytes or total lymph node cells. Total thymocyte numbers are provided in the histograms. Results are shown for two independently generated {epsilon}M-tg founder lines with similar transgene copy number.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 3. (A) Functional responses of T cells from CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. CD4+CD8- T cells were purified from lymph nodes and cultured on plates precoated with anti-CD3{epsilon} and varying amounts of anti-CD28 mAbs as indicated. After 40 h, cells were pulsed with [3H]thymidine and harvested. {blacksquare}, CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg. {blacktriangleup}, CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg. Results shown are representative of those obtained in five of six experiments (in one experiment, the proliferative responses of T cells from CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice were not significantly different). (B) Analysis of proliferative responses by CFSE labeling. Experimental conditions were identical to those described in A except that cells were labeled with CFSE before stimulation. Histograms show CFSE staining on live gated CD4+ cells. Results shown are representative of three individual experiments. (C) Proliferative responses of CD8-SP thymocytes from H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. CD4-CD8+ thymocytes were purified and cultured on plates with APCs prepared from female mice + peptide antigen (Smcy; KCSRNRQYL). After 40 h, cells were pulsed with [3H]thymidine and harvested. {blacksquare}, H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg. {blacktriangleup}, H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg. {diamondsuit}, CD3{epsilon}+/+.


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

T Cell Development Is Restored in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg Mice.
T cell development is arrested at the CD4-CD8- (DN) stage in CD3{epsilon}{Delta}/{Delta} mice, such that the more mature subsets of CD4+CD8+ (DP), CD4+CD8- (CD4-SP), and CD4-CD8+ (CD8-SP) thymocytes normally present in the thymus of adult mice are absent in CD3{epsilon}{Delta}/{Delta} mice (Fig 1 A; reference 15). As a consequence of this developmental block, total thymocyte numbers are markedly reduced in CD3{epsilon}{Delta}/{Delta} mice (<10% of normal), and mature T cells are undetectable in the peripheral lymphoid organs (Fig 1 A; reference 15). As reported previously (15) and shown in Fig 1 A, expression of transgenic WT CD3{epsilon} ({epsilon}-tg) in CD3{epsilon}{Delta}/{Delta} mice can rescue this developmental block, as assessed by the normalization of thymus cellularity and the presence of normal numbers of DP thymocytes and mature, TCRhi SP T cells in the thymus and periphery. Significantly, genetic reconstitution with a transgene encoding a mutant form of CD3{epsilon} that contains a Y->F mutation of the distal tyrosine residue within the single CD3{epsilon} ITAM ({epsilon}M-tg) also rescued the developmental defect in CD3{epsilon}{Delta}/{Delta} mice (Fig 1 A). CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice contained similar numbers of thymocytes, and these were composed of approximately the same ratio of DP, CD4-SP, and CD8-SP cells as control, CD3{epsilon}+/+ mice (Fig 1 A). In addition, normal numbers of TCRhi CD4-SP and TCRhi CD8-SP T cells were present in the periphery of CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice (Fig 1 B).

The normalization of thymus cellularity in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice also indicated that the mutant CD3{epsilon} protein could restore surface expression of pre-TCR complexes on immature DN thymocytes and that these complexes could transduce signals sufficient to promote the transition of DN thymocytes to the DP stage. To explore this issue further, we examined the phenotype of DN thymocytes obtained from CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. As shown previously (15) and in Fig 1 C, DN thymocytes are arrested at the CD44- CD25+ stage in CD3{epsilon}{Delta}/{Delta} mice. The observation that mutations in several distinct genes that are required either for pre-TCR surface expression or for pre-TCR signaling result in developmental arrest at this identical stage indicates that signaling by the pre-TCR is necessary for the maturation of DN, CD44-CD25+ thymocytes to the CD44- CD25- stage (1). Notably, DN, CD44-CD25- thymocytes were present in {epsilon}{Delta}/{Delta}; {epsilon}M-tg mice in numbers similar to those observed in CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg mice (Fig 1 C), indicating that functional pre-TCR complexes are expressed in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. Finally, the mutant CD3{epsilon} chain was also capable of rescuing the development of DN-{alpha} TCR+ and DN {gamma}/{delta} TCR+ T cells when introduced into CD3{epsilon}{Delta}/{Delta} mice, as these cells were detected both in the thymus (Fig 1C) and periphery (data not shown) of CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice.

The {epsilon}M Transgene Encodes a Signaling-defective Protein.
Based on the results of in vitro studies, the {epsilon}M transgene is predicted to encode a protein that does not contribute to the TCR signaling response (4) (5) (20) (21). However, in view of the normal phenotype observed in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice, we wished to examine this issue in greater detail in our transgenic system. A hallmark of ITAM function is its ability to become tyrosine phosphorylated upon T cell activation, thereby creating a site for stable association with and activation of ZAP-70 (2). After activation with pervanadate, tyrosine phosphorylation of both CD3{epsilon} and TCR-{zeta} is observed in thymocytes from control (CD3{epsilon}+/+) and CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg mice (Fig 2 A). In contrast, whereas pervanadate stimulation of thymocytes from CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice induced tyrosine phosphorylation of TCR {zeta} chain, tyrosine phosphorylation of the mutated CD3{epsilon} chain was nearly undetectable (Fig 2 A). Similar results were obtained after stimulation of thymocytes by TCR cross-linking (data not shown). Reblotting with anti-CD3{epsilon} mAb demonstrated the presence of the mutant CD3{epsilon} protein in the immunoprecipitated CD3 complexes (Fig 2 A). As the proximal ITAM tyrosine is retained in the {epsilon}M protein, the extremely low level of tyrosine phosphorylation observed after stimulation was unexpected. Interestingly, a TCR {zeta} chain transgene containing Y->F mutations of the NH2- (but not the COOH-) terminal tyrosine residues in each of its three ITAMs also failed to become phosphorylated in vivo upon T cell activation (9). Recent data suggest that tyrosine phosphorylation of the TCR {zeta} chain follows a hierarchical progression in which the phosphorylation of specific ITAM tyrosines proceeds and perhaps facilitates phosphorylation of other ITAM tyrosines (22). Our findings may reflect a similar hierarchy for phosphorylation of the CD3{epsilon}-ITAM tyrosines. In any event, these results indicate that the {epsilon}M protein is weakly (if at all) tyrosine phosphorylated upon TCR engagement.

Although a defect in thymocyte maturation was not observed in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice, we reasoned that one might be revealed in the presence of another mutation that impairs pre-TCR/TCR surface expression and/or signaling. Mice lacking TCR {zeta} chain ({zeta}-/-) exhibit a profound, though incomplete, block in T cell development characterized by reduced TCR surface expression and a reduction in the number of DP and SP thymocytes and mature, peripheral SP T cells (reference (23); Fig 2 B). The low number of DP thymocytes in {zeta}-/- mice indicates that pre-TCR surface expression and/or pre-TCR signaling is compromised in the absence of TCR {zeta} chain. When mice rendered deficient for both CD3{epsilon} and TCR-{zeta} ({epsilon}{Delta}/{Delta} x {zeta}-/-) were reconstituted with the WT CD3{epsilon} transgene, a phenotype essentially indistinguishable from that of {zeta}-/- mice was observed (i.e., low numbers of DP and SP thymocytes and peripheral T cells), reflecting the ability of the transgenic CD3{epsilon} protein to rescue the {epsilon}{Delta}/{Delta} but not the {zeta}-/- defect (Fig 2 B). However, two independently generated {epsilon}M transgenes expressing similar (~1x) levels of {epsilon}M protein were unable to effect a similar partial rescue of T cell development when introduced into {epsilon}{Delta}/{Delta} x {zeta}-/- mice (Fig 2 B). In addition to demonstrating that the {epsilon}M protein is signaling defective in vivo, these results reveal that a signaling threshold exists for the pre-TCR, below which this complex is incapable of inducing the formation of DP thymocytes. These findings are consistent with those of Ardouin et al. (24) demonstrating a gene dosage effect of CD3{epsilon} for pre-TCR function in the absence of {zeta} chain. As both pre-TCR surface expression and signaling are thought to be compromised in {zeta}-/- mice, a more complete understanding of pre-TCR signaling requirements awaits the analysis of {epsilon}{Delta}/{Delta} x {zeta}-/- mice reconstituted with signaling-deficient forms of both proteins.

TCR Signaling Responses Are Intact or Mildly Impaired in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg Mice.
To assess whether the TCR complexes expressed on thymocytes from CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice can transduce signals similar to those normally elicited by TCR cross-linking, we first examined if TCR engagement led to the activation of downstream signaling pathways. Several early events in the TCR activation cascade, such as the phosphorylation of {zeta} chain (Fig 2 A), ZAP-70, and LAT (linker for activation of T cells; data not shown), were unaffected in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. These results were not unexpected, as ZAP-70 is known to be activated following its association with phosphorylated {zeta} chain ITAMs after TCR engagement, and {zeta} chain expression is unaltered in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. Activation-induced calcium responses were also indistinguishable in thymocytes from CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice (data not shown).

Mature T cell responses in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice were examined by analyzing the ability of peripheral T cells to proliferate and produce IL-2 after cross-linking of the TCR and CD28. Although no significant differences were observed under conditions of maximal TCR stimulation, T cells from CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice exhibited impaired proliferative responses (Fig 3 A) and cytokine responses (data not shown) when limiting concentrations of stimulating antibody were used (Fig 3 A). Impaired proliferative responses were also observed when proliferation was assessed by CFSE staining (Fig 3 B). Together, these results demonstrate that although CD3{epsilon}-mediated signals are not specifically required for T cell maturation or mature T cell functional responses, they contribute quantitatively to TCR signaling.

Thymocyte Selection and T Cell Survival in TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg Mice.
To examine the role of CD3{epsilon} signals in thymocyte selection, we bred the H-Y TCR transgene into the CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg backgrounds. Positive selection of thymocytes in H-Y TCR-tg/H-2Db female mice results in the generation of CD8-SP thymocytes and T cells that express high levels of the H-Y clonotypic TCR (detected by mAb T3.70) (16). Positive selection of H-Y TCR+ thymocytes appeared uncompromised in H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice when compared with H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg mice, as assessed by total thymocyte numbers and the number and percentage of CD8-SP/T3.70hi cells in the thymus (Fig 4 A). However, examination of peripheral lymphoid organs revealed a striking paucity of CD8-SP/T3.70hi T cells in H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice (Fig 4 A). Low numbers of CD8-SP/T3.70hi T cells were consistently observed in both the lymph nodes and spleens of H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice regardless of the age of the mice tested (range 4 wk to 6 mo). Although CD8-SP/T3.70hi T cell numbers were markedly reduced in H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice, CD8-SP/T3.70- and CD4-SP T cell numbers were similar to or greater than those in H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg mice, indicating that the generation and/or survival of CD8-SP/T3.70hi peripheral T cells was selectively compromised in H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice.




View larger version (41K):
[in this window]
[in a new window]
 
Figure 4. Positive selection of thymocytes from CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice expressing H-Y– or P14-transgenic TCRs. (A) Comparison of thymocytes and lymph node T cells from H-Y TCR–transgenic/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and H-Y TCR-transgenic/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg females. Two-color plots show CD4/CD8 staining profiles on total or T3.70+ (gated) thymocytes and lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show staining of total thymocytes or CD8+ lymph node T cells with the H-Y clonotype–specific antibody, T3.70. (The percentage of T3.70hi cells and the gate used for the T3.70+ two-color plots is also shown.) Results are representative of four separate experiments. (B) Comparison of thymocytes and lymph node T cells from P14 TCR–transgenic/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and P14 TCR-transgenic/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice. Two-color plots show CD4/CD8 staining profiles on total or V{alpha}2+ (gated) thymocytes and lymph node cells. Numbers in quadrants indicate the percentage of cells within that quadrant. Histograms show staining of total thymocytes or CD8+ lymph node T cells with the P14 TCR {alpha} chain–reactive mAb V{alpha}2. (The percentage of V{alpha}2hi cells and the gate used for the V{alpha}2+ two-color plots is also shown.) Results are representative of two separate experiments.

One possible explanation for the results obtained with H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice is that T3.70hi CD8-SP thymocytes fail to complete the maturation process in the thymus. However, arguing against this, H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg and H-Y TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice contained similar numbers of phenotypically (Fig 4 and data not shown) and functionally (Fig 3 C) mature T3.70hi CD8-SP thymocytes.

To determine if the effect of the {epsilon}M-tg was specific to cells that express the H-Y TCR, we examined development and selection of thymocytes expressing a different MHC class I–restricted TCR, P14. Thymocytes that express the P14 TCR are also positively selected in the H-2Db background, leading to the generation of CD8-SP/V{alpha}2hi thymocytes and peripheral T cells (17). Examination of thymocytes and peripheral (lymph node) T cells from P14 TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice revealed a phenotype nearly identical to that of P14 TCR-tg/CD3{epsilon}{Delta}/{Delta}; {epsilon}-tg mice (i.e., mice of both genotypes contained similar numbers of CD8-SP/V{alpha}2hi thymocytes and peripheral T cells) (Fig 4 B). Thus, the generation and survival of CD8-SP T cells that express the P14 TCR did not appear to be compromised in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice.

Although other explanations remain possible, these results are most consistent with the notion that CD3{epsilon} signals are required for the survival of naive H-Y TCR+ but not P14 TCR+ T cells. Recent studies have shown that self-MHC is required for long-term survival of naive T cells (25) (26). However, to our knowledge, the data presented here are the first that directly implicate TCR signaling in T cell survival. Our results could reflect a unique requirement for CD3{epsilon}-mediated signals in T survival; however, they are also consistent with the idea that CD3{epsilon} contributes quantitatively to the TCR signaling response. A generalized defect in T cell survival was not observed in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice but instead was seen only in the H-Y TCR–transgenic background. Significantly, the self-ligand that positively selects and presumably maintains the survival of peripheral H-Y TCR+ T cells is thought to bind with especially low affinity to the TCR (27) (28). Thus, the survival of H-Y TCR+ CD8-SP T cells but not P14 TCR+ CD8-SP T cells may critically depend upon the presence of the entire complement of TCR-ITAMs. Consistent with this idea, the generation of H-Y TCR+ CD8-SP T cells but not P14 TCR+ CD8-SP T cells by positive selection was found to be absolutely dependent upon TCR {zeta} chain ITAMs (9) (10). Moreover, P14 TCR+ CD8-SP T cells but not H-Y TCR+ CD8-SP T cells are triggered to undergo homeostatic proliferation when transferred to H-2Db T cell–depleted hosts, again suggesting that H-Y TCR+ CD8-SP T cells bind with lower affinity to self-ligands (28) (29). Another possibility that cannot presently be ruled out is that the mutant CD3{epsilon} chain expressed in CD3{epsilon}{Delta}/{Delta}; {epsilon}M-tg mice is not inert but instead inhibits TCR signaling. An inhibitory role for {zeta} chains that contain partially (mono)phosphorylated ITAMs has been postulated based on data from in vitro studies (30). However, an inhibitory effect was not observed when a {zeta} chain containing mutated ITAMs capable of becoming mono- but not diphosphorylated was expressed in transgenic mice (9). Additional experiments will be required to determine the precise function of TCR signaling in general and CD3{epsilon} signals in particular in regulating T cell survival.


  Footnotes

Connie L. Sommers' present address is Laboratory of Cellular and Molecular Biology, Division of Basic Sciences, National Cancer Institute, Bethesda, MD 20892-4255.


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

We thank Dr. Ronald Germain for critical reading of the manuscript and Victoria Saunders for technical assistance.

Submitted: 4 May 2000
Revised: 23 June 2000
Accepted: 12 July 2000


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

  1. Von Boehmer, H., and Fehling, H.J. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol 15:433-452[Medline].

  2. Wange, R.L., and Samelson, L.E. 1996. Complex complexes: signaling at the TCR. Immunity 5:197-205[Medline].

  3. Sebzda, E., Mariathasan, S., Ohteki, T., Jones, R., Bachmann, M.F., and Ohashi, P.S. 1999. Selection of the T cell repertoire. Annu. Rev. Immunol 17:829-874[Medline].

  4. Isakov, N., Wange, R.L., Burgess, W.H., Watts, J.D., Aebersold, R., and Samelson, L.E. 1995. ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity. J. Exp. Med. 181:375-380[Abstract].

  5. Osman, N., Turner, H., Lucas, S., Reif, K., and Cantrell, D.A. 1996. The protein interactions of the immunoglobulin receptor family tyrosine-based activation motifs present in the T cell receptor {zeta} subunits and the CD3 {gamma}{delta}{epsilon} chains. Eur. J. Immunol 26:1063-1068[Medline].

  6. Romeo, C., Amiot, M., and Seed, B. 1992. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor {zeta}-chain. Cell 68:889-897[Medline].

  7. Irving, B.A., Chan, A.C., and Weiss, A. 1993. Functional characterization of a signal transducing motif present in the T cell antigen receptor zeta chain. J. Exp. Med 177:1093-1103[Abstract].

  8. Shores, E.W., Huang, K., Tran, T., Lee, E., Grinberg, A., and Love, P.E. 1994. Role of TCR-{zeta} chain in T cell development and selection. Science. 266:1047-1050[Medline].

  9. Ardouin, L., Boyer, C., Gillet, A., Trucy, J., Bernard, A.-M., Nunes, J., Delon, J., Trautmann, A., He, H.-T., and Malissen, B. et al. 1999. Crippling of CD3-{zeta} ITAMs does not impair T cell receptor signaling. Immunity. 10:409-420[Medline].

  10. Shores, E.W., Tran, T., Grinberg, A., Sommers, C.L., Shen, H., and Love, P.E. 1997. Role of the multiple T cell receptor TCR-{zeta} chain signaling motifs in selection of the T cell repertoire. J. Exp. Med. 185:893-900[Abstract/Free Full Text].

  11. Shinkai, Y., Ma, A., Cheng, H.-L., and Alt, F.W. 1995. CD3{epsilon} and CD3{zeta} cytoplasmic domains can independently generate signals for T cell development and function. Immunity 2:401-411[Medline].

  12. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E., and Malissen, B. 1995. Altered T cell development in mice with a targeted mutation of the CD3-{epsilon} gene. EMBO (Eur. Mol. Biol. Organ.) J. 14:4641-4653[Abstract].

  13. Dave, V.P., Cao, Z., Browne, C., Alarcon, B., Fernandez-Miguel, G., Lafaille, J., de la Hera, A., Tonegawa, S., and Kappes, D.J. 1997. CD3{delta} deficiency arrests development of the {alpha}ß but not the {gamma}{delta} T cell lineage. EMBO (Eur. Mol. Biol. Organ.) J. 16:1360-1370[Abstract/Free Full Text].

  14. Haks, M.C., Krimpenfort, P., Borst, J., and Kruisbeek, A.M. 1998. The CD3{gamma} chain is essential for development of both the TCR{alpha}ß and TCR{gamma}{delta} lineages. EMBO (Eur. Mol. Biol. Organ.) J. 17:1871-1882[Free Full Text].

  15. DeJarnette, J., Sommers, C.L., Huang, K., Woodside, K.J., Emmons, R., Katz, K., Shores, E.W., and Love, P.E. 1998. Specific requirement for CD3, in T cell development. Proc. Natl. Acad. Sci. USA. 95:14909-14914[Abstract/Free Full Text].

  16. von Boehmer, H. 1990. Developmental biology of T cells in T cell receptor transgenic mice. Annu. Rev. Immunol 8:531-556[Medline].

  17. Pircher, H., Bürki, K., Lang, R., Hengartner, H., and Zinkernagel, R.M. 1989. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen. Nature. 342:559-561[Medline].

  18. Van Oers, N., Love, P.E., Shores, E.W., and Weiss, A. 1998. Regulation of TCR signal transduction in thymocytes by multiple TCR-{zeta} chain signaling motifs. J. Immunol 160:163-170[Abstract/Free Full Text].

  19. Sommers, C.L., Rabin, R.L., Grinberg, A., Tsay, H.C., Farber, J., and Love, P.E. 1999. A role for the Tec family kinase Txk in T cell activation and thymocyte selection. J. Exp. Med. 190:1427-1438[Abstract/Free Full Text].

  20. Bu, J.Y., Shaw, A.S., and Chan, A.C. 1995. Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance. Proc. Natl. Acad. Sci. USA. 92:5106-5110[Abstract].

  21. de Aos, I., Metzger, M.H., Exley, M., Dahl, C.E., Misra, S., Zheng, D., Varticovski, L., Terhorst, C., and Sancho, J. 1997. Tyrosine phosphorylation of the CD3-{epsilon} subunit of the T cell antigen receptor mediates enhanced association with phosphatidylinositol 3-kinase in Jurkat T cells. J. Biol. Chem. 272:25310-25318[Abstract/Free Full Text].

  22. Kersh, E.N., Shaw, A.S., and Allen, P.M. 1998. Fidelity of T cell activation through multistep T cell receptor zeta phosphorylation. Science. 281:572-575[Abstract/Free Full Text].

  23. Love, P.E., Shores, E.W., Johnson, M.D., Tremblay, M., Lee, E.J., Grinberg, A., Huang, S.P., Singer, A., and Westphal, H. 1993. T cell development in mice that lack the {zeta} chain of the T cell antigen receptor complex. Science. 261:918-921[Medline].

  24. Ardouin, L., Ismaili, J., Malissen, B., and Malissen, M. 1998. The CD3-{gamma}{delta}{epsilon} and CD3-{zeta}/{eta} modules are each essential for allelic exclusion at the T cell receptor ß locus but are both dispensable for the initiation of V to(D)J recombination at the T cell receptor-ß, -{gamma}, and -{delta} loci. J. Exp. Med. 187:105-116[Abstract/Free Full Text].

  25. Nesic, D., and Vukmanovic, S. 1998. MHC class I is required for peripheral accumulation of CD8+ thymic emigrants. J. Immunol. 160:3705-3712[Abstract/Free Full Text].

  26. Brocker, T. 1997. Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex II–expressing dendritic cells. J. Exp. Med. 186:1223-1232[Abstract/Free Full Text].

  27. Robey, E.A., Fowlkes, B.J., Gordon, J.W., Kioussis, D., von Boehmer, H., Ramsdell, F., and Axel, R. 1991. Thymic selection in CD8 transgenic mice supports an instructive model for commitment to a CD4 or CD8 lineage. Cell 64:99-109[Medline].

  28. Ernst, B., Lee, D.-S., Chang, J.M., Sprent, J., and Surh, C.D. 1999. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 11:173-181[Medline].

  29. Tanchot, C., Lemonnier, F.A., Perarnau, B., Freitas, A.A., and Rocha, B. 1997. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science 276:2057-2062[Abstract/Free Full Text].

  30. Kersh, E.N., Kersh, G.J., and Allen, P.M. 1999. Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation. J. Exp. Med 190:1627-1636.