Stoichiometry of the murine T cell receptor
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《实验药学杂志》
Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
CORRESPONDENCE Sandra M. Hayes: hayessa@upstate.edu
The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCR chains. In most vertebrates, there are two different clonotypic heterodimers (TCR? and TCR) that define the ? and T cell lineages, respectively. ?- and TCRs also differ in their invariant chain subunit composition, in that ?TCRs contain CD3 and CD3 dimers, whereas TCRs contain only CD3 dimers. This difference in subunit composition of the ?- and TCRs raises the question of whether the stoichiometries of these receptor complexes are different. As the stoichiometry of the murine TCR has not been previously investigated, we used two quantitative immunofluorescent approaches to determine the valency of TCR heterodimers and CD3 dimers in surface murine TCR complexes. Our results support a model of murine TCR stoichiometry in which there are two CD3 dimers for every TCR heterodimer.
The multimeric TCR is composed of an antigen-binding clonotypic heterodimer (TCR? or TCR) and a signal-transducing complex, consisting of the CD3 dimers (CD3 and/or CD3) and a TCR homodimer. TCR signaling is required for lineage commitment and repertoire selection during development, for maintenance of the peripheral T cell pool, and for differentiation of naive T cells into effector and memory cell populations during an immune response. Despite the fact that many of the components of the TCR-coupled signaling pathways have been elucidated, it is not precisely known how these signaling events are initially triggered. Two models have been proposed to provide a mechanism for the initiation of TCR signal transduction, with each implicating a distinct stoichiometry for the TCR (for reviews see references 1 and 2). In the first model, the surface TCR complex contains one TCR heterodimer, two CD3 dimers, and one TCR homodimer (3–6). According to this monovalent TCR model, ligand engagement would initiate signaling by conformational changes in the subunits and/or by oligomerization of individual TCR complexes. The second model, known as the bivalent TCR model, proposes that the surface TCR complex contains two clonotypic heterodimers, two CD3 dimers, and one TCR homodimer (7–10). Triggering of the bivalent TCR complex may still require a conformational change in the subunits, but the presence of two TCR heterodimers within a single TCR complex would preclude the need for receptor oligomerization.
Most studies of TCR stoichiometry have been performed on the ?TCR and, consequently, little is known about the stoichiometry of TCR. We recently provided evidence to suggest that the stoichiometry of the TCR differs from that of the ?TCR. Specifically, whereas ?TCRs contain both CD3 and CD3 dimers, most TCRs were found to contain only CD3 dimers (11). However, these experiments did not resolve whether one or two CD3 dimers are incorporated into the fully assembled TCR complex. Interestingly, signal transduction by the TCR was shown to be superior to that of the ?TCR after cross-linking of CD3 alone (11). A multivalent TCR complex could explain this enhanced signaling capacity of the TCR. To determine the stoichiometry of the TCR, we developed two quantitative immunofluorescence techniques to measure (a) the ratio of CD3 dimers to TCR heterodimers and (b) the relative percentage of CD3 dimers on the surface of polyclonal T cells. In this paper, we report findings that favor a monovalent model for TCR stoichiometry.
RESULTS AND DISCUSSION
Quantifying the ratio of CD3 dimers to TCR heterodimers on the surface of murine T cells
There are three possible models for the stoichiometry of the murine TCR. Two of these (Fig. 1, A and B) are based on the present models proposed for the stoichiometry of the ?TCR. The configuration in Fig. 1 A is based on the monovalent ?TCR model (3–6) and depicts the surface TCR complex with one TCR heterodimer, two CD3 dimers, and one TCR homodimer, for a total of eight subunits. The configuration in Fig. 1 B is based on the alternative bivalent ?TCR model (7–10) and depicts the surface TCR complex with 2 TCR heterodimers, 2 CD3 dimers, and 1 TCR homodimer, for a total of 10 subunits. It is also conceivable that the rules of TCR assembly and surface expression differ from those of the ?TCR, such that a TCR complex containing only one CD3 dimer is transported to and stably expressed on the cell surface. This surface complex would contain one TCR heterodimer, one CD3 dimer, and one TCR homodimer, for a total of six subunits (Fig. 1 C). As the CD3/TCR ratio varies in the configurations shown in Fig. 1, quantifying this ratio is the first step in solving the stoichiometry of the murine TCR. To this end, we developed a flow cytometric approach similar to those used by others to quantify the CD3/TCR ratio on primary mouse and human T cells (4, 12). This method takes advantage of the fact that the mAbs against CD3/ dimers (2C11) and TCR heterodimers (GL3, GL4, UC7-13D5, and UC3-10A6) are all hamster IgG antibodies containing light chains. As each primary antibody can be detected with the same anti–hamster Ig secondary antibody, the relative expression levels of CD3 dimers and TCR heterodimers on the surface of T cells can be measured if saturating amounts of mAb are used. Our approach differs from those of previous studies in that we used a monoclonal anti–hamster antibody instead of polyclonal anti–hamster IgG antibodies, thereby restricting recognition to a single epitope on each primary antibody. A representative staining profile for anti-CD3/ (2C11) and two anti-TCR (GL3 and UC7-13D5) mAbs on gated CD4–CD8–CD19– LN cells from TCR?–/– mice is shown in Fig. 2 A. Note that the relative fluorescence of 2C11 mAb staining is approximately twice that of anti-TCR mAb staining, regardless of which anti-TCR mAb was used (GL3, GL4, UC7-13D5, or UC3-10A6; Fig. 2, A and B). These results indicate that there are two CD3 dimers for every TCR heterodimer on the surface of T cells. Importantly, loss of CD3 expression does not affect this ratio, because we also observed two CD3 dimers for every TCR heterodimer on the surface of CD4–CD8– TCR?–CD19– LN cells from CD3–/– mice (Fig. 2 B). This finding is consistent with previous results demonstrating that neither TCR nor TCR pairs efficiently to a CD3 dimer (11). The observed 2:1 ratio of CD3 dimers to TCR heterodimers favors the monovalent TCR model shown in Fig. 1 A, in which there is one TCR heterodimer and two CD3 dimers in each TCR complex. Thus, our findings indicate that the TCR has a signal transducing complex that is similar to that of the ?TCR, in that it contains two CD3 dimers.
Figure 1. Possible configurations for the murine TCR complex. (A) Monovalent TCR model. (B) Bivalent TCR model. (C) One CD3dimer model.
Figure 2. Comparison of CD3 and TCR surface levels on murine T cells. (A) Histogram showing the relative fluorescence of 2C11, GL3, and UC7-13D5 (UC7) mAb staining on gated CD4–CD8–CD19– LN cells from TCR?–/– mice. The 2C11 mAb (dark gray line) recognizes both CD3 and CD3 dimers, and the GL3 (bold line) and UC7 (light gray line) mAbs recognize TCR heterodimers. Staining with a hamster isotype control is also shown (shaded histogram). The number in parentheses represents the mean fluorescence intensity for each mAb minus that of the hamster isotype control. (B) Ratio of CD3/TCR (for calculation information see Materials and methods) for each anti-TCR mAb on the surface of CD4–CD8–CD19– LN cells from TCR?–/– mice (GL3, n = 10; GL4, n = 3; UC7, n = 10; UC3, n = 3) and CD4–CD8–TCR?–CD19– LN cells from CD3–/– mice (GL3, n = 8; GL4, n = 4; UC7, n = 8; UC3, n = 3). Bars represent the means ± standard deviation.
Quantifying the relative percentage of CD3 dimers on the surface of murine T cells
Biochemical analysis suggests that a small percentage of surface TCR complexes contain CD3 dimers (11, 13). The TCRs that contain CD3 dimers could be restricted to a distinct subpopulation of T cells or may represent a minor subset of TCRs on each T cell. To discern between these two possibilities, we developed a second flow cytometric assay that uses an anti-CD3 mAb (7D6), which has been reported to block the binding of the 2C11 mAb to CD3 dimers but not to CD3 dimers (14). To confirm the specificity of the 7D6 mAb and its ability to block 2C11 mAb staining of CD3 dimers, we assayed double-negative (DN) thymocytes from CD3–/–, CD3–/–, and CD3–/– mice (Fig. 3 A). The 7D6 mAb detected the intracellular CD3 dimers present in CD3–/– DN thymocytes but not the intracellular CD3 dimers present in CD3–/– DN thymocytes. In addition, pretreatment with the 7D6 mAb completely blocked 2C11 mAb staining of intracellular CD3 dimers in CD3–/– DN thymocytes but had no effect on 2C11 mAb staining of intracellular CD3 dimers in CD3–/– DN thymocytes. Next, we tested the efficacy of this flow cytometric approach. We first assayed T cells from CD3–/– mice, which express only CD3 dimers on their cell surface (13), and found that the 7D6 mAb was indeed able to completely block 2C11 mAb surface staining (Fig. 3 B). We then assayed CD4+ ? T cells from B6 mice, which express both CD3 and CD3 dimers on their cell surface (for review see reference 15) and found, as expected, that the 7D6 mAb only partially blocked 2C11 mAb surface staining (Fig. 3 B). If expression of TCRs containing CD3 dimers were limited to a subpopulation of T cells, then pretreatment with purified 7D6 mAb should partially block 2C11 staining on this CD3+ subset and completely block 2C11 staining on the CD3– subset. However, if CD3 containing TCRs were a minor subset of TCRs expressed on each T cell, then pretreatment with purified 7D6 mAb should almost completely block 2C11 staining on all T cells. We found that when T cells from TCR?–/– mice were assayed, the 7D6 mAb almost completely blocked the staining of the 2C11 mAb, indicating that the TCRs containing CD3 dimers represent a minor subset of TCRs expressed on each T cell (Fig. 3 B). In fact, the relative percentage of CD3 dimers was calculated to be 99.2 ± 0.1% of all CD3 dimers.
Figure 3. Quantifying the relative percentage of CD3 dimers on the surface of murine T cells. (A) Intracellular staining (ic) of CD3 dimers in gated DN thymocytes from CD3–/– (bold line) and CD3–/– mice (gray line) using FITC-conjugated 2C11 (anti-CD3/) and Alexa 488–conjugated 7D6 (anti-CD3) mAbs. DN thymocytes are defined as CD4–CD8–CD19–TCR–TCR?–NK1.1–DX5–. Gated DN thymocytes from CD3–/– mice were used as a negative staining control (shaded histogram). Staining without (top) and with (bottom) 7D6 mAb blocking is shown. (B) Surface (s) staining of CD3 dimers on gated TCR+ CD4–CD8– LN cells from CD3–/– and TCR?–/– mice and on gated ?TCR+ CD4+ LN cells from B6 mice using 2C11–FITC and 7D6–Alexa 488 mAbs. (top) Surface staining of 7D6–Alexa 488 mAb alone (bold line) and after blocking with purified 7D6 mAb (shaded histogram). (bottom) Surface staining of 2C11–FITC mAb alone (gray line), after blocking with purified 7D6 mAb (bold line), and after blocking with purified 2C11 mAb (shaded histogram). The mean relative percentage of CD3 dimers on TCR+ CD4–CD8– LN cells from TCR?–/– mice (n = 4; for calculation see Materials and methods) ± standard deviation is shown.
CD3 is absolutely required for the assembly and expression of the TCR
If two CD3 dimers are found in each surface TCR complex, then the loss of CD3 should have profound effects on TCR assembly and surface expression. Indeed, Haks et al. have reported that T cell development is severely affected in CD3–/– mice (16). We sought to expand these earlier experiments by performing a more detailed analysis of TCR surface expression on thymocytes and splenocytes from CD3–/– mice and from CD3–/– mice carrying a TCR transgene. Virtually no TCR+ cells were detected in the thymus and spleen of CD3–/– mice (Fig. 4, A and B). Moreover, introduction of a TCR transgene into CD3–/– mice did not increase the number of TCR+ thymocytes and splenocytes as it does when introduced into CD3+/+ mice, indicating that the absence of TCR+ cells in CD3–/– mice cannot solely be caused by a failure to express productively rearranged TCR and - genes (Fig. 4 A). These findings demonstrate that TCR assembly and surface expression are absolutely dependent on the presence of CD3. Remarkably, unlike the TCR, the ?TCR can still be expressed on the surface of CD3–/– thymocytes and splenocytes, albeit at reduced levels compared with CD3+/+ cells (Fig. 4 C) (16). Therefore, CD3–/– mice reveal a difference in the requirement for CD3 in ?- and TCR assembly and surface expression. Importantly, this difference is consistent with the supposition that TCR and - chains each pair with a CD3 dimer but not a CD3 dimer.
Figure 4. Phenotypic analysis of CD3–/– and TCR Tg CD3–/– mice. (A) Comparison of TCR surface levels on DN thymocytes and splenocytes from CD3–/– (bold line) and CD3+/+ (gray line) mice (top) and from TCR Tg CD3–/– (bold line) and TCR Tg CD3+/+ (gray line) mice (bottom). DN thymocytes and splenocytes are defined as CD4–CD8–CD19–. Staining with a hamster isotype control is also shown (shaded histogram). (B) Number of TCR+ CD4–CD8–CD19– cells in the thymus and spleen of CD3+/+ (n = 4) and CD3–/– (n = 6) mice. Bars represent means ± standard deviation. (C) Comparison of ?TCR surface levels on CD4+ thymocytes and splenocytes from CD3+/+ (gray line) and CD3–/– (bold line) mice. Staining with a hamster isotype control is also shown (shaded histogram).
Concluding remarks
Using quantitative immunofluorescence techniques, we have addressed the issue of murine TCR stoichiometry. We observed a 2:1 ratio of CD3 dimers to TCR heterodimers on the surface of peripheral T cells, a ratio that supports the monovalent TCR model (Fig. 1 A). We also present new evidence, in accordance with previously reported biochemical data (11, 13), demonstrating that the two CD3 dimers contained within the TCR are almost exclusively CD3 dimers. Lastly, an analysis of TCR surface expression on CD3–/– thymocytes and splenocytes revealed an absolute requirement for CD3 dimers in TCR assembly. Together, these data strongly support the idea that, during TCR assembly, both TCR and TCR pair with a CD3 dimer. In this study, the ratio of TCR homodimers to CD3 dimers or TCR heterodimers was not measured and, therefore, the number of TCR homodimers contained within a surface TCR complex cannot be determined. However, based on the conservation of positively charged residues in the transmembrane regions of all four TCR chains that are required for association with the invariant TCR chains (for review see reference 15), we propose that, like the ?TCR, the TCR contains one TCR homodimer.
The vast majority of murine TCRs, whether expressed on naive or activated T cells, contain only CD3 dimers (Fig. 3 and not depicted) (11, 13). It is not clear, however, whether human TCRs share the same bias for CD3 dimers. Biochemical analysis of surface TCR complexes on primary human T cells detected some CD3 dimers but considerably less than the amount observed in surface ?TCR complexes on primary human ? T cells (11). Interestingly, biochemical analysis of surface TCR complexes on activated and expanded human T cell clones detected CD3 dimers in amounts comparable to those seen in surface ?TCRs (unpublished data) (17). Unfortunately, CD3 deficiency in humans does not resolve the issue of whether CD3 is required for human TCR surface expression, because it is not known whether the absence of peripheral T cells (18, 19) is caused by the loss of CD3 or by the markedly reduced levels of the other invariant subunits that accompany CD3 deficiency in the patients analyzed (18). Nevertheless, these findings suggest that there may be important differences in the subunit requirements for murine and human TCR assembly. It is believed, based on sequence homology, that TCR is the counterpart to TCR and, consequently, that TCR should pair preferentially with CD3 dimers (for review see reference 15). Accordingly, the inconsistency in murine and human TCR assembly can be explained by a difference in either the binding affinities of the respective TCR chains for CD3 dimers or the binding affinities of the respective CD3 dimers for TCR chains. Murine TCR pairs to a CD3 dimer but not to a CD3 dimer (Figs. 2 and 3) (11, 13). However, this is not the case for human TCR, as a metabolic labeling study using TCR?– Jurkat cells transfected with a human TCR gene shows that human TCR can associate with either human CD3 or CD3 (20). Remarkably, in the same study, when a murine TCR gene was transfected into the TCR?– Jurkat variant, the murine TCR was also shown to pair with either human CD3 or CD3. Collectively, these data indicate that murine and human CD3 dimers differ in their ability to bind to TCR chains.
Of the current models of TCR stoichiometry, the observed 2:1 ratio of CD3 dimers to TCR heterodimers favors the monovalent TCR model for TCR stoichiometry (Fig. 1 A). However, we cannot rule out the possibility that monovalent TCR complexes cluster or aggregate on the cell surface to form higher order complexes. If these higher order complexes exist, they may provide an explanation for how signal transduction by the TCR is superior to that of the ?TCR in the absence of coreceptor involvement. The difference in the subunit composition of the ?- and TCR signal transducing complexes may also explain the increased signaling proficiency of the TCR. As the amino acid sequence of the immunoreceptor tyrosine-based activation motif in each CD3 chain is unique (for review see reference 21), it is conceivable that ?- and TCR complexes recruit distinct signaling molecules. In addition, or alternatively, intrinsic differences in the signaling pathways coupled to ?- and TCRs may provide a mechanism by which the TCR is capable of signaling better than the ?TCR.
MATERIALS AND METHODS
Mice.
B6.129P2-TCR?–/– (TCR?–/–) mice (22) were purchased from the Jackson Laboratory. C57BL/6-CD3–/– (CD3–/–) mice (23) were provided by D. Kappes (Fox Chase Cancer Center, Philadelphia, PA), and 129-CD3–/– (CD3–/–) mice (16) were provided by D. Wiest (Fox Chase Cancer Center). C57BL/6-V6/V1 TCR transgenic (Tg) mice (line 134) (24) were provided by B.J. Fowlkes (National Institutes of Health , Bethesda, MD). B6.129-CD3–/– (CD3–/–) (25) and C57BL/6 (B6) mice were generated in our colony. Mice were bred and maintained in an NIH Research Animal Facility in accordance with the specifications of the Association for Assessment and Accreditation of Laboratory Animal Care. Mouse protocols were approved by the NIH Animal Care and Use Committee. All mice were killed at 8–12 wk of age.
Antibodies and reagents.
mAbs used for flow cytometric analysis included anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-TCR (GL3, GL4, and UC7-13D5), anti-V4 (UC3-10A6), anti-TCR? (H57-597), anti-CD3 (145-2C11), anti-CD19 (1D3), and a hamster IgG isotype control, all of which were purchased from BD Biosciences. The secondary reagent, biotin-conjugated anti–hamster Ig (HIG-29), was also purchased from BD Biosciences. The anti-CD3 (7D6) hybridoma (14) was obtained from A. Singer (NIH, Bethesda, MD) and D. Wiest and was used to produce ascites. Protein A/G–purified 7D6 mAb was conjugated to AlexaFluor 488 according to the manufacturer's instructions (Invitrogen). AlexaFluor 488 conjugated to streptavidin was also purchased from Invitrogen.
Flow cytometry.
Flow cytometric analysis for surface antigens was performed as previously described (26). Intracellular staining for CD3 dimers was performed (Cytofix/Cytoperm kit; BD Biosciences) according to the manufacturer's instructions. The ratio of CD3 dimers to TCR heterodimers was determined using an assay previously described for determining the ratio of CD3 dimers to TCR? heterodimers (4, 12). In brief, 1.5 x 106 lymph node cells were incubated with saturating amounts of purified anti-TCR (GL3, GL4, UC7-13D5, or UC3-10A6) and anti-CD3 (145-2C11) mAbs for 30 min on ice. Saturating amounts of antibody are defined as the concentration of purified antibody required to completely block the binding of the same antibody conjugated to a fluorochrome. All five mAbs are hamster IgG that use the Ig light chain. Accordingly, their relative binding intensities can be assayed using saturating amounts of the same anti–hamster secondary mAb, biotin-conjugated anti–hamster Ig (HIG-29). The CD3/TCR ratio was calculated using the following equation, where MFI stands for mean fluorescence intensity:
The relative percentage of CD3 dimers in CD3 dimers on the surface of T cells was determined using saturating amounts of anti-CD3 (7D6; 300 μg/ml for intracellular staining and 200 μg/ml for surface staining) mAb to block the binding of CD3 dimers by anti-CD3 (145-2C11) mAb (14). The percentage of CD3 dimers was calculated using the following equation:
For all experiments, 2–4 x 105 cells were collected (FACSCalibur; Becton Dickinson) using CellQuest software or LSR II using FACSDiva software (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded from analysis based on forward and side scatter profiles.
Acknowledgments
We thank Dalal El-Khoury for excellent technical assistance. We also thank Drs. Dietmar Kappes, B.J. Fowlkes, and David Wiest for mice, and Drs. Alfred Singer and David Wiest for the 7D6 hybridoma.
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CORRESPONDENCE Sandra M. Hayes: hayessa@upstate.edu
The T cell receptor for antigen (TCR) complex is organized into two functional domains: the antigen-binding clonotypic heterodimer and the signal-transducing invariant CD3 and TCR chains. In most vertebrates, there are two different clonotypic heterodimers (TCR? and TCR) that define the ? and T cell lineages, respectively. ?- and TCRs also differ in their invariant chain subunit composition, in that ?TCRs contain CD3 and CD3 dimers, whereas TCRs contain only CD3 dimers. This difference in subunit composition of the ?- and TCRs raises the question of whether the stoichiometries of these receptor complexes are different. As the stoichiometry of the murine TCR has not been previously investigated, we used two quantitative immunofluorescent approaches to determine the valency of TCR heterodimers and CD3 dimers in surface murine TCR complexes. Our results support a model of murine TCR stoichiometry in which there are two CD3 dimers for every TCR heterodimer.
The multimeric TCR is composed of an antigen-binding clonotypic heterodimer (TCR? or TCR) and a signal-transducing complex, consisting of the CD3 dimers (CD3 and/or CD3) and a TCR homodimer. TCR signaling is required for lineage commitment and repertoire selection during development, for maintenance of the peripheral T cell pool, and for differentiation of naive T cells into effector and memory cell populations during an immune response. Despite the fact that many of the components of the TCR-coupled signaling pathways have been elucidated, it is not precisely known how these signaling events are initially triggered. Two models have been proposed to provide a mechanism for the initiation of TCR signal transduction, with each implicating a distinct stoichiometry for the TCR (for reviews see references 1 and 2). In the first model, the surface TCR complex contains one TCR heterodimer, two CD3 dimers, and one TCR homodimer (3–6). According to this monovalent TCR model, ligand engagement would initiate signaling by conformational changes in the subunits and/or by oligomerization of individual TCR complexes. The second model, known as the bivalent TCR model, proposes that the surface TCR complex contains two clonotypic heterodimers, two CD3 dimers, and one TCR homodimer (7–10). Triggering of the bivalent TCR complex may still require a conformational change in the subunits, but the presence of two TCR heterodimers within a single TCR complex would preclude the need for receptor oligomerization.
Most studies of TCR stoichiometry have been performed on the ?TCR and, consequently, little is known about the stoichiometry of TCR. We recently provided evidence to suggest that the stoichiometry of the TCR differs from that of the ?TCR. Specifically, whereas ?TCRs contain both CD3 and CD3 dimers, most TCRs were found to contain only CD3 dimers (11). However, these experiments did not resolve whether one or two CD3 dimers are incorporated into the fully assembled TCR complex. Interestingly, signal transduction by the TCR was shown to be superior to that of the ?TCR after cross-linking of CD3 alone (11). A multivalent TCR complex could explain this enhanced signaling capacity of the TCR. To determine the stoichiometry of the TCR, we developed two quantitative immunofluorescence techniques to measure (a) the ratio of CD3 dimers to TCR heterodimers and (b) the relative percentage of CD3 dimers on the surface of polyclonal T cells. In this paper, we report findings that favor a monovalent model for TCR stoichiometry.
RESULTS AND DISCUSSION
Quantifying the ratio of CD3 dimers to TCR heterodimers on the surface of murine T cells
There are three possible models for the stoichiometry of the murine TCR. Two of these (Fig. 1, A and B) are based on the present models proposed for the stoichiometry of the ?TCR. The configuration in Fig. 1 A is based on the monovalent ?TCR model (3–6) and depicts the surface TCR complex with one TCR heterodimer, two CD3 dimers, and one TCR homodimer, for a total of eight subunits. The configuration in Fig. 1 B is based on the alternative bivalent ?TCR model (7–10) and depicts the surface TCR complex with 2 TCR heterodimers, 2 CD3 dimers, and 1 TCR homodimer, for a total of 10 subunits. It is also conceivable that the rules of TCR assembly and surface expression differ from those of the ?TCR, such that a TCR complex containing only one CD3 dimer is transported to and stably expressed on the cell surface. This surface complex would contain one TCR heterodimer, one CD3 dimer, and one TCR homodimer, for a total of six subunits (Fig. 1 C). As the CD3/TCR ratio varies in the configurations shown in Fig. 1, quantifying this ratio is the first step in solving the stoichiometry of the murine TCR. To this end, we developed a flow cytometric approach similar to those used by others to quantify the CD3/TCR ratio on primary mouse and human T cells (4, 12). This method takes advantage of the fact that the mAbs against CD3/ dimers (2C11) and TCR heterodimers (GL3, GL4, UC7-13D5, and UC3-10A6) are all hamster IgG antibodies containing light chains. As each primary antibody can be detected with the same anti–hamster Ig secondary antibody, the relative expression levels of CD3 dimers and TCR heterodimers on the surface of T cells can be measured if saturating amounts of mAb are used. Our approach differs from those of previous studies in that we used a monoclonal anti–hamster antibody instead of polyclonal anti–hamster IgG antibodies, thereby restricting recognition to a single epitope on each primary antibody. A representative staining profile for anti-CD3/ (2C11) and two anti-TCR (GL3 and UC7-13D5) mAbs on gated CD4–CD8–CD19– LN cells from TCR?–/– mice is shown in Fig. 2 A. Note that the relative fluorescence of 2C11 mAb staining is approximately twice that of anti-TCR mAb staining, regardless of which anti-TCR mAb was used (GL3, GL4, UC7-13D5, or UC3-10A6; Fig. 2, A and B). These results indicate that there are two CD3 dimers for every TCR heterodimer on the surface of T cells. Importantly, loss of CD3 expression does not affect this ratio, because we also observed two CD3 dimers for every TCR heterodimer on the surface of CD4–CD8– TCR?–CD19– LN cells from CD3–/– mice (Fig. 2 B). This finding is consistent with previous results demonstrating that neither TCR nor TCR pairs efficiently to a CD3 dimer (11). The observed 2:1 ratio of CD3 dimers to TCR heterodimers favors the monovalent TCR model shown in Fig. 1 A, in which there is one TCR heterodimer and two CD3 dimers in each TCR complex. Thus, our findings indicate that the TCR has a signal transducing complex that is similar to that of the ?TCR, in that it contains two CD3 dimers.
Figure 1. Possible configurations for the murine TCR complex. (A) Monovalent TCR model. (B) Bivalent TCR model. (C) One CD3dimer model.
Figure 2. Comparison of CD3 and TCR surface levels on murine T cells. (A) Histogram showing the relative fluorescence of 2C11, GL3, and UC7-13D5 (UC7) mAb staining on gated CD4–CD8–CD19– LN cells from TCR?–/– mice. The 2C11 mAb (dark gray line) recognizes both CD3 and CD3 dimers, and the GL3 (bold line) and UC7 (light gray line) mAbs recognize TCR heterodimers. Staining with a hamster isotype control is also shown (shaded histogram). The number in parentheses represents the mean fluorescence intensity for each mAb minus that of the hamster isotype control. (B) Ratio of CD3/TCR (for calculation information see Materials and methods) for each anti-TCR mAb on the surface of CD4–CD8–CD19– LN cells from TCR?–/– mice (GL3, n = 10; GL4, n = 3; UC7, n = 10; UC3, n = 3) and CD4–CD8–TCR?–CD19– LN cells from CD3–/– mice (GL3, n = 8; GL4, n = 4; UC7, n = 8; UC3, n = 3). Bars represent the means ± standard deviation.
Quantifying the relative percentage of CD3 dimers on the surface of murine T cells
Biochemical analysis suggests that a small percentage of surface TCR complexes contain CD3 dimers (11, 13). The TCRs that contain CD3 dimers could be restricted to a distinct subpopulation of T cells or may represent a minor subset of TCRs on each T cell. To discern between these two possibilities, we developed a second flow cytometric assay that uses an anti-CD3 mAb (7D6), which has been reported to block the binding of the 2C11 mAb to CD3 dimers but not to CD3 dimers (14). To confirm the specificity of the 7D6 mAb and its ability to block 2C11 mAb staining of CD3 dimers, we assayed double-negative (DN) thymocytes from CD3–/–, CD3–/–, and CD3–/– mice (Fig. 3 A). The 7D6 mAb detected the intracellular CD3 dimers present in CD3–/– DN thymocytes but not the intracellular CD3 dimers present in CD3–/– DN thymocytes. In addition, pretreatment with the 7D6 mAb completely blocked 2C11 mAb staining of intracellular CD3 dimers in CD3–/– DN thymocytes but had no effect on 2C11 mAb staining of intracellular CD3 dimers in CD3–/– DN thymocytes. Next, we tested the efficacy of this flow cytometric approach. We first assayed T cells from CD3–/– mice, which express only CD3 dimers on their cell surface (13), and found that the 7D6 mAb was indeed able to completely block 2C11 mAb surface staining (Fig. 3 B). We then assayed CD4+ ? T cells from B6 mice, which express both CD3 and CD3 dimers on their cell surface (for review see reference 15) and found, as expected, that the 7D6 mAb only partially blocked 2C11 mAb surface staining (Fig. 3 B). If expression of TCRs containing CD3 dimers were limited to a subpopulation of T cells, then pretreatment with purified 7D6 mAb should partially block 2C11 staining on this CD3+ subset and completely block 2C11 staining on the CD3– subset. However, if CD3 containing TCRs were a minor subset of TCRs expressed on each T cell, then pretreatment with purified 7D6 mAb should almost completely block 2C11 staining on all T cells. We found that when T cells from TCR?–/– mice were assayed, the 7D6 mAb almost completely blocked the staining of the 2C11 mAb, indicating that the TCRs containing CD3 dimers represent a minor subset of TCRs expressed on each T cell (Fig. 3 B). In fact, the relative percentage of CD3 dimers was calculated to be 99.2 ± 0.1% of all CD3 dimers.
Figure 3. Quantifying the relative percentage of CD3 dimers on the surface of murine T cells. (A) Intracellular staining (ic) of CD3 dimers in gated DN thymocytes from CD3–/– (bold line) and CD3–/– mice (gray line) using FITC-conjugated 2C11 (anti-CD3/) and Alexa 488–conjugated 7D6 (anti-CD3) mAbs. DN thymocytes are defined as CD4–CD8–CD19–TCR–TCR?–NK1.1–DX5–. Gated DN thymocytes from CD3–/– mice were used as a negative staining control (shaded histogram). Staining without (top) and with (bottom) 7D6 mAb blocking is shown. (B) Surface (s) staining of CD3 dimers on gated TCR+ CD4–CD8– LN cells from CD3–/– and TCR?–/– mice and on gated ?TCR+ CD4+ LN cells from B6 mice using 2C11–FITC and 7D6–Alexa 488 mAbs. (top) Surface staining of 7D6–Alexa 488 mAb alone (bold line) and after blocking with purified 7D6 mAb (shaded histogram). (bottom) Surface staining of 2C11–FITC mAb alone (gray line), after blocking with purified 7D6 mAb (bold line), and after blocking with purified 2C11 mAb (shaded histogram). The mean relative percentage of CD3 dimers on TCR+ CD4–CD8– LN cells from TCR?–/– mice (n = 4; for calculation see Materials and methods) ± standard deviation is shown.
CD3 is absolutely required for the assembly and expression of the TCR
If two CD3 dimers are found in each surface TCR complex, then the loss of CD3 should have profound effects on TCR assembly and surface expression. Indeed, Haks et al. have reported that T cell development is severely affected in CD3–/– mice (16). We sought to expand these earlier experiments by performing a more detailed analysis of TCR surface expression on thymocytes and splenocytes from CD3–/– mice and from CD3–/– mice carrying a TCR transgene. Virtually no TCR+ cells were detected in the thymus and spleen of CD3–/– mice (Fig. 4, A and B). Moreover, introduction of a TCR transgene into CD3–/– mice did not increase the number of TCR+ thymocytes and splenocytes as it does when introduced into CD3+/+ mice, indicating that the absence of TCR+ cells in CD3–/– mice cannot solely be caused by a failure to express productively rearranged TCR and - genes (Fig. 4 A). These findings demonstrate that TCR assembly and surface expression are absolutely dependent on the presence of CD3. Remarkably, unlike the TCR, the ?TCR can still be expressed on the surface of CD3–/– thymocytes and splenocytes, albeit at reduced levels compared with CD3+/+ cells (Fig. 4 C) (16). Therefore, CD3–/– mice reveal a difference in the requirement for CD3 in ?- and TCR assembly and surface expression. Importantly, this difference is consistent with the supposition that TCR and - chains each pair with a CD3 dimer but not a CD3 dimer.
Figure 4. Phenotypic analysis of CD3–/– and TCR Tg CD3–/– mice. (A) Comparison of TCR surface levels on DN thymocytes and splenocytes from CD3–/– (bold line) and CD3+/+ (gray line) mice (top) and from TCR Tg CD3–/– (bold line) and TCR Tg CD3+/+ (gray line) mice (bottom). DN thymocytes and splenocytes are defined as CD4–CD8–CD19–. Staining with a hamster isotype control is also shown (shaded histogram). (B) Number of TCR+ CD4–CD8–CD19– cells in the thymus and spleen of CD3+/+ (n = 4) and CD3–/– (n = 6) mice. Bars represent means ± standard deviation. (C) Comparison of ?TCR surface levels on CD4+ thymocytes and splenocytes from CD3+/+ (gray line) and CD3–/– (bold line) mice. Staining with a hamster isotype control is also shown (shaded histogram).
Concluding remarks
Using quantitative immunofluorescence techniques, we have addressed the issue of murine TCR stoichiometry. We observed a 2:1 ratio of CD3 dimers to TCR heterodimers on the surface of peripheral T cells, a ratio that supports the monovalent TCR model (Fig. 1 A). We also present new evidence, in accordance with previously reported biochemical data (11, 13), demonstrating that the two CD3 dimers contained within the TCR are almost exclusively CD3 dimers. Lastly, an analysis of TCR surface expression on CD3–/– thymocytes and splenocytes revealed an absolute requirement for CD3 dimers in TCR assembly. Together, these data strongly support the idea that, during TCR assembly, both TCR and TCR pair with a CD3 dimer. In this study, the ratio of TCR homodimers to CD3 dimers or TCR heterodimers was not measured and, therefore, the number of TCR homodimers contained within a surface TCR complex cannot be determined. However, based on the conservation of positively charged residues in the transmembrane regions of all four TCR chains that are required for association with the invariant TCR chains (for review see reference 15), we propose that, like the ?TCR, the TCR contains one TCR homodimer.
The vast majority of murine TCRs, whether expressed on naive or activated T cells, contain only CD3 dimers (Fig. 3 and not depicted) (11, 13). It is not clear, however, whether human TCRs share the same bias for CD3 dimers. Biochemical analysis of surface TCR complexes on primary human T cells detected some CD3 dimers but considerably less than the amount observed in surface ?TCR complexes on primary human ? T cells (11). Interestingly, biochemical analysis of surface TCR complexes on activated and expanded human T cell clones detected CD3 dimers in amounts comparable to those seen in surface ?TCRs (unpublished data) (17). Unfortunately, CD3 deficiency in humans does not resolve the issue of whether CD3 is required for human TCR surface expression, because it is not known whether the absence of peripheral T cells (18, 19) is caused by the loss of CD3 or by the markedly reduced levels of the other invariant subunits that accompany CD3 deficiency in the patients analyzed (18). Nevertheless, these findings suggest that there may be important differences in the subunit requirements for murine and human TCR assembly. It is believed, based on sequence homology, that TCR is the counterpart to TCR and, consequently, that TCR should pair preferentially with CD3 dimers (for review see reference 15). Accordingly, the inconsistency in murine and human TCR assembly can be explained by a difference in either the binding affinities of the respective TCR chains for CD3 dimers or the binding affinities of the respective CD3 dimers for TCR chains. Murine TCR pairs to a CD3 dimer but not to a CD3 dimer (Figs. 2 and 3) (11, 13). However, this is not the case for human TCR, as a metabolic labeling study using TCR?– Jurkat cells transfected with a human TCR gene shows that human TCR can associate with either human CD3 or CD3 (20). Remarkably, in the same study, when a murine TCR gene was transfected into the TCR?– Jurkat variant, the murine TCR was also shown to pair with either human CD3 or CD3. Collectively, these data indicate that murine and human CD3 dimers differ in their ability to bind to TCR chains.
Of the current models of TCR stoichiometry, the observed 2:1 ratio of CD3 dimers to TCR heterodimers favors the monovalent TCR model for TCR stoichiometry (Fig. 1 A). However, we cannot rule out the possibility that monovalent TCR complexes cluster or aggregate on the cell surface to form higher order complexes. If these higher order complexes exist, they may provide an explanation for how signal transduction by the TCR is superior to that of the ?TCR in the absence of coreceptor involvement. The difference in the subunit composition of the ?- and TCR signal transducing complexes may also explain the increased signaling proficiency of the TCR. As the amino acid sequence of the immunoreceptor tyrosine-based activation motif in each CD3 chain is unique (for review see reference 21), it is conceivable that ?- and TCR complexes recruit distinct signaling molecules. In addition, or alternatively, intrinsic differences in the signaling pathways coupled to ?- and TCRs may provide a mechanism by which the TCR is capable of signaling better than the ?TCR.
MATERIALS AND METHODS
Mice.
B6.129P2-TCR?–/– (TCR?–/–) mice (22) were purchased from the Jackson Laboratory. C57BL/6-CD3–/– (CD3–/–) mice (23) were provided by D. Kappes (Fox Chase Cancer Center, Philadelphia, PA), and 129-CD3–/– (CD3–/–) mice (16) were provided by D. Wiest (Fox Chase Cancer Center). C57BL/6-V6/V1 TCR transgenic (Tg) mice (line 134) (24) were provided by B.J. Fowlkes (National Institutes of Health , Bethesda, MD). B6.129-CD3–/– (CD3–/–) (25) and C57BL/6 (B6) mice were generated in our colony. Mice were bred and maintained in an NIH Research Animal Facility in accordance with the specifications of the Association for Assessment and Accreditation of Laboratory Animal Care. Mouse protocols were approved by the NIH Animal Care and Use Committee. All mice were killed at 8–12 wk of age.
Antibodies and reagents.
mAbs used for flow cytometric analysis included anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-TCR (GL3, GL4, and UC7-13D5), anti-V4 (UC3-10A6), anti-TCR? (H57-597), anti-CD3 (145-2C11), anti-CD19 (1D3), and a hamster IgG isotype control, all of which were purchased from BD Biosciences. The secondary reagent, biotin-conjugated anti–hamster Ig (HIG-29), was also purchased from BD Biosciences. The anti-CD3 (7D6) hybridoma (14) was obtained from A. Singer (NIH, Bethesda, MD) and D. Wiest and was used to produce ascites. Protein A/G–purified 7D6 mAb was conjugated to AlexaFluor 488 according to the manufacturer's instructions (Invitrogen). AlexaFluor 488 conjugated to streptavidin was also purchased from Invitrogen.
Flow cytometry.
Flow cytometric analysis for surface antigens was performed as previously described (26). Intracellular staining for CD3 dimers was performed (Cytofix/Cytoperm kit; BD Biosciences) according to the manufacturer's instructions. The ratio of CD3 dimers to TCR heterodimers was determined using an assay previously described for determining the ratio of CD3 dimers to TCR? heterodimers (4, 12). In brief, 1.5 x 106 lymph node cells were incubated with saturating amounts of purified anti-TCR (GL3, GL4, UC7-13D5, or UC3-10A6) and anti-CD3 (145-2C11) mAbs for 30 min on ice. Saturating amounts of antibody are defined as the concentration of purified antibody required to completely block the binding of the same antibody conjugated to a fluorochrome. All five mAbs are hamster IgG that use the Ig light chain. Accordingly, their relative binding intensities can be assayed using saturating amounts of the same anti–hamster secondary mAb, biotin-conjugated anti–hamster Ig (HIG-29). The CD3/TCR ratio was calculated using the following equation, where MFI stands for mean fluorescence intensity:
The relative percentage of CD3 dimers in CD3 dimers on the surface of T cells was determined using saturating amounts of anti-CD3 (7D6; 300 μg/ml for intracellular staining and 200 μg/ml for surface staining) mAb to block the binding of CD3 dimers by anti-CD3 (145-2C11) mAb (14). The percentage of CD3 dimers was calculated using the following equation:
For all experiments, 2–4 x 105 cells were collected (FACSCalibur; Becton Dickinson) using CellQuest software or LSR II using FACSDiva software (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded from analysis based on forward and side scatter profiles.
Acknowledgments
We thank Dalal El-Khoury for excellent technical assistance. We also thank Drs. Dietmar Kappes, B.J. Fowlkes, and David Wiest for mice, and Drs. Alfred Singer and David Wiest for the 7D6 hybridoma.
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