Reversal of the TCR Stop Signal by CTLA-4

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Science  29 Sep 2006:
Vol. 313, Issue 5795, pp. 1972-1975
DOI: 10.1126/science.1131078


The coreceptor cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is pivotal in regulating the threshold of signals during T cell activation, although the underlying mechanism is still not fully understood. Using in vitro migration assays and in vivo two-photon laser scanning microscopy, we showed that CTLA-4 increases T cell motility and overrides the T cell receptor (TCR)–induced stop signal required for stable conjugate formation between T cells and antigen-presenting cells. This event led to reduced contact periods between T cells and antigen-presenting cells that in turn decreased cytokine production and proliferation. These results suggest a fundamentally different model of reverse stop signaling, by which CTLA-4 modulates the threshold for T cell activation and protects against autoimmunity.

The T cell coreceptor CTLA-4 has a powerful modulatory effect on the signals that induce T cell cytokine production and proliferation (1, 2). Consequently, CTLA-4–deficient (CTLA-4–/–) mice show a profound postthymic autoimmune phenotype marked by massive tissue infiltration and organ destruction (3, 4). Proposed mechanisms for the negative influence of CTLA-4 include ectodomain competition for binding of the related costimulatory molecule CD28 to CD80/86 (5), disruption of CD28 localization at the immunological synapse (IS) (6), modulation of TCR signaling by protein phosphatase 2A and the tyrosine phosphatase SHP-2 (79), and interference with the expression or composition of lipid rafts on the surface of T cells (1012). In addition, CTLA-4 engagement of CD80/86 on dendritic cells (DCs) can induce the release of indoleamine 2,3-dioxygenase (13), whereas CD4+CD25+ regulatory T cells can modulate disease in the CTLA-4–/– mouse (1416). Antibodies to CTLA-4 (anti–CTLA-4) and CTLA-4–immunoglobulin have been used as therapeutics in the modulation of autoimmunity, transplantation, and tumor immunotherapy (17, 18).

CTLA-4 is a potent direct activator and a TCR coactivator of LFA-1 integrin clustering and adhesion (19). To assess whether CTLA-4 could also influence integrin-dependent motility, we tracked preactivated CTLA-4+/+ and CTLA-4–/– T cells for movement on plates coated with the LFA-1 ligand intercellular adhesion molecule–1 (ICAM-1) (Fig. 1). In contrast to untreated CTLA-4+/+ and CTLA-4–/– cells, which migrated at similar speeds (Fig. 1, A, C, and E), CTLA-4+/+ cells treated with anti–CTLA-4 increased motility (Fig. 1, B versus A). As anticipated, the effects of anti–CTLA-4 were not observed with CTLA-4–/– T cells (Fig. 1, D versus C; E). Thus, in addition to increasing LFA-1 adhesion (19), CTLA-4 readily increased the movement of T cells.

Fig. 1.

CTLA-4 ligation increases T cell motility. Preactivated primary CD4+ Tcells from CTLA-4+/+ (A and B) and CTLA-4–/– (C and D) mice were treated with anti–CTLA-4 and assessed for motility on ICAM-1–coated plates as described (23, 35). WT, wild type; KO, knockout. (E) Boxplots showing the motility of cells. Solid circles indicate means. n.s., not significant. The star indicates a statistical difference based on an analysis of variance (ANOVA).

TCR ligation reduces or arrests T cell motility (i.e., the stop signal), an event that is required for stable T cell conjugate formation with antigen-presenting cells (APCs) and efficient activation (2022). Given that CTLA-4 increased T cell motility, we assessed whether the coreceptor could override this stop signal. Preactivated murine and human T cells were monitored for 20 min after exposure to anti-CD3 in the presence or absence of anti–CTLA-4. In contrast to the ligation of anti-CD3, which reduced the movement of mouse and human primary T cells (Fig. 2, B versus A and F versus E), the coligation of CTLA-4 and anti-CD3 reversed the arrest such that treated and untreated cells moved at similar speeds (Fig. 2, C versus A and G versus E; D and H). Similar effects were seen with the CTLA-4–transfected T cell hybridoma DC27.10 (fig. S1). These findings showed that CTLA-4 can override the stop signal induced by the antigen receptor complex.

Fig. 2.

(A to D) CTLA-4 ligation reverses the TCR-induced stop signal of mouse primary T cells. Preactivated primary CD4+ Tcells were stimulated with anti-CD3 or anti–CD3/CTLA-4 and assessed for motility on ICAM-1–coated plates as described (23, 35). (A) media control; (B) anti-CD3; (C) anti-CD3/CTLA-4; (D) boxplots of motility patterns. Solid circles indicate means. a, medium; b, anti-CD3; c, anti–CD3/CTLA-4. The three stars represent a statistical difference based on ANOVA. (E to H) CTLA-4 ligation reverses the TCR-induced stop signal of human primary T cells. Preactivated human peripheral T cells were stimulated with anti-CD3 or anti–CD3/CTLA-4 and were monitored for movement as described (23). (E) media control; (F) anti-CD3; (G) anti–CD3/CTLA-4; (H) boxplots of motility patterns. Solid circles indicate means. a, medium; b, anti-CD3; c, anti–CD3/CTLA-4. The three asterisks indicate a statistical difference based on ANOVA.

To extend our findings to an in vivo setting, we separated preactivated CTLA-4 positive and negative CD4+ T cells transgenic for the DO11.10 TCR using beads coated with anti–CTLA-4 (23). These cells were then labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked in cervical lymph nodes by two-photon laser scanning microscopy (TPLSM) (Fig. 3). T cells in mice injected with antigen have been shown to undergo changes in motility involving initial transient serial encounters, followed by a phase of slowing and stable contacts and then by a return to high motility (22, 2427). In our study, CTLA-4 positive and negative T cells were monitored at 15, 20, and 24 hours after injection with ovalbumin (OVA) peptide. The first noticeable difference between cells was a more extended, polarized appearance of CTLA-4 positive cells (fig. S2 and movies S1 to S4). In the absence of peptide, CTLA-4–expressing and –nonexpressing T cells showed a random walk pattern with similar motilities (Fig. 3, A and E versus G). After the OVA peptide injection, CTLA-4 negative cells showed the anticipated decrease in mean motility (Fig. 3, A and F versus E). By contrast, the motility of CTLA-4 positive cells remained unchanged and occasionally increased in the presence of OVA peptide (Fig. 3, A and H versus F; fig. S2, movies S1 to S4). The three-dimensional motility coefficient showed a reduction from 140 to 29.7 μm3 min–1 for CTLA-4 negative cells and an increase from 114 to 210 μm3 min–1 for CTLA-4 positive cells (Fig. 3B). Dot plot analysis showed some heterogeneity in the motility of individual cells, although the mean motility of CTLA-4 positive cells remained unchanged in the presence of peptide (Fig. 3, I and J). Despite this result, the two subsets had similar volumes (Fig. 3C) and meandering indices (Fig. 3D). This resistance of CTLA-4 positive cells to antigen-induced slowing was observed at all times examined. These experiments revealed that CTLA-4 positive cells fail to stop or slow down in response to the in vivo peptide challenge.

Fig. 3.

Two-photon laser-scanning microscopy of CTLA-4 positive and negative DO11.10 T cells shows that expressing cells fail to stop in response to OVA peptide. Isolated CTLA-4 positive and negative D011.10 T cells were injected through the tail vein, and then phosphate-buffered saline or chicken OVA peptide was injected as described (23, 27). Excised cervical lymph nodes were imaged essentially as described (27). (A to D) Measurement of in vivo T cell behavior by TPLSM shows that CTLA-4 positive T cells do not slow down in response to peptide. Error bars represent SEM. Data in movies S1 to S4 were analyzed with PicViewer and Volocity software. (A) velocity; (B) motility coefficient; (C) volumes; (D) meandering factor. (E to H) Representative movement of CTLA-4 positive and negative cells observed by TPLSM. The different color traces represent single cells. The behavior of T cells in the presence and absence of peptide antigen was monitored with Volocity software. (E) CTLA-4 recipients with no OVA; (F) CTLA-4 recipients with OVA; (G) CTLA-4+ recipients with no OVA; (H) CTLA-4+ recipients with OVA. (I and J) Dot blot analysis of individual CTLA-4 positive and negative cells in response to peptide. The tracing of individual T cells showed heterogeneity with a reduction in the median velocity (μm min–1) and motility coefficient (μm3 min–1) of CTLA-4 negative cells in response to injected peptide, whereas CTLA-4 positive cells failed to reduce motility. Horizontal bars indicate SEM.

Because the slowing of motility is required for stable T cell–APC conjugate formation (2022, 26, 27), the reversal of the stop signal by CTLA-4 suggested that the coreceptor might reduce the period of interaction. To test this conjecture, we incubated CTLA-4 positive and negative DO11.10 T cells with a CFSE-labeled B cell line, A20 (Fig. 4A), or labeled mature DCs (Fig. 4B). APCs were left untreated or were preincubated with OVA peptide. Individual T cells were then monitored for the duration of APC binding over a 1200-s period. Whereas CTLA-4 negative T cells incubated with APCs without peptide showed short-term interactions (75% of which were less than 400 s), the addition of OVA peptide induced longer term interactions (60 to 70% were more than 600 s). In contrast, CTLA-4 positive T cells failed to form long-term interactions even in the presence of antigen. Instead, like those cells unexposed to peptide, CTLA-4 positive cells under-took short-term interactions. We observed this effect when A20 B cells or DCs were used as APCs (Fig. 4, A and B). In turn, these shorter dwell periods (Fig. 4C) resulted in lower interleukin-2 (IL-2) production (Fig. 4D) and proliferation (Fig. 4E). CTLA-4 expression therefore led to more transient interactions with APCs and a reduction in T cell activation.

Fig. 4.

CTLA-4 positive T cells show more transient conjugation periods and a concurrent reduction in IL-2 production and proliferation. Purified preactivated DO11.10 CD4+ T cells were then incubated with A20 B cells or mature DCs preloaded with OVA peptide as described (35). (A and B) Comparison of the conjugation times of CTLA-4 positive and negative cells in the presence and absence of OVA peptide. Whereas the conjugation time of CTLA-4 negative cells was extended in response to added peptide, the conjugation time of CTLA-4 positive cells was unaffected. A20 B cells and mature DCs were used as APCs in (A) and (B), respectively. Horizontal bars indicate SEM. (C to E) Reduced conjugation periods of CTLA-4 positive cells correlate with reduced levels of IL-2 production and proliferation. IL-2 production and proliferation were measured as described (19). Representative experiments showing differences in the period of conjugation (C), level of IL-2 production (D), and proliferation (E) for CTLA-4 negative and positive cells. Error bars in (D) and (E) represent SEM. cpm, counts per minute.

Overall, our observations support a reverse stop-signaling mechanism for modulating the threshold of T cell activation by CTLA-4, which is distinct from previous models of CTLA-4 function. By limiting T cell–APC contact times, CTLA-4 would reduce the efficiency of the major histocompatibility complex (MHC)–peptide presentation and the number of TCR ligation events resulting in reduced signaling and activation. Shorter dwell times could also alter receptor rearrangements at the IS and account for the reduced localization of CD28 (6). In this way, CTLA-4 may be tailored to regulate the secondary responses of effectors such as cytotoxic T lymphocytes, whose shorter dwell times are sufficient to elicit cytotoxicity. More transient interactions may in turn increase the frequency of contacts between effector and target cells. CTLA-4 positive cells may also compete with CTLA-4 negative cells to inhibit longer term conjugation with APCs and facilitate a rapid exit from lymph nodes and reentry to the circulation for migration to sites of inflammation.

The role of CTLA-4 as a gatekeeper of conjugation may also account for the connection with autoimmunity (3, 4). Reduced conjugation might protect against prolonged contact periods that allow for responses to lower-affinity autoantigen. Increased LFA-1–mediated adhesion in the absence of increased motility allows for responses to low-affinity subthreshold agonist including self-MHC molecules (2830). This may also explain the reported requirement for anergy induction of the T cell (31, 32). In certain settings, by limiting dwell times, suboptimal or altered signaling may result in nonresponsiveness. Indeed, tolerance induction is accompanied by less stable DC–T cell interactions (26, 33), smaller clusters (27), and a rapid restoration of motility (33). Regulatory T cells can also limit T cell–APC contact times (34). Further studies will more precisely elucidate the role of altered motility and conjugation in the full range of functions regulated by the coreceptor.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

Movies S1 to S4

References and Notes

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