The identification of small molecules that alter T cell interactions with antigen presenting cells represents an intriguing therapeutic strategy for autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Indeed, a recent study has highlighted the critical importance of T cell and APC contact duration in determining T cell fate in vivo
and the development of T cell tolerance or activation (9
). There are currently no known small molecules which reverse the T cell stop signal in clinical use, and the addition of such drugs to treat autoimmune diseases is particularly attractive given the high cost of biologic agents and the resultant burden on the healthcare system. Here, we have identified at least three distinct classes of “reverse-stop” small molecules that impair TCR-induced T cell arrest but not random T cell motility: 1) Src family tyrosine kinase inhibitors, 2) microtubule depolymerizing agents, and 3) prostaglandins. These compounds act in contrast to inhibitors of phospholipase C (U73122), which block both basal and activated T cell motility or sphingosine-1-phosphate analog FTY720 and the PI3K inhibitor LY-294002 which altered basal motility but did not affect adhesion or spreading induced by OKT3 (Supplemental Table S3
The requirement of Src family kinases for the TCR induced T cell stop signal but not for T cell random motility indicates that Src inhibitors represent T cell stop signal antagonists. This is consistent with the model that proximal T cell signaling is necessary for TCR-induced T cell arrest. It is intriguing that not all Src kinase inhibitors, most notably SKII, are capable of reversing the T cell stop signal. The results suggest that the stop signal is dependent on a Src family kinase which is preferentially targeted by PP1, PP2, and SU6656 but not SKI1.
Previous work has indicated that Src kinase activation is required for TCR-mediated polarization of the microtubule organizing center toward the T cell-APC contact (26
). It is interesting that our data indicate that Src inhibitors and microtubule disruption impair T cell stopping and interactions with APC. This is, to our knowledge, the first report to show that microtubules are necessary for the T cell arrest induced by TCR ligation. In accordance with our findings, previous studies have reported that microtubule disruption induces random motility of neutrophils (28
) and modulates T cell random migration through rho/ROCK signaling (29
). However, ROCK inhibition did not affect TCR-induced T cell stopping in our system, suggesting that effects of microtubule inhibition on T cell arrest may be independent of Rho/ROCK signaling.
The finding that both microtubule polymerization inhibitors and prostaglandins are capable of preventing the T cell stop signal without affecting ZAP-70 or LAT phosphorylation (i.e. proximal T cell receptor signaling) is particularly interesting (). In fact, we had initially hypothesized that the screening results would yield molecules that work to disrupt proximal signaling, such as the Src inhibitors. Our findings suggest that it is possible to decouple proximal T cell receptor signaling from the T cell receptor stop signal. PGE2 had no effect on the phosphorylation of Lck or Fyn at concentrations that block T cell arrest, suggesting that PGE2 effects on T cell arrest are independent of its effects on Src kinase activity. Our findings identified a novel role for PGE2 in regulation of the small GTPase Rap1, which is critical for TCR induced inside out activation of LFA-1.
To our knowledge, this is the first report to implicate PGE2 in regulating the T cell stop signal. In contrast, previous studies have reported that PGE2 stimulates the ability of DCs to induce T cell proliferation (30
). The finding that PGE1 and PGE2 impair T cell migration stopping as well as inhibit T cell proliferation (31
) indicate there may be counteracting mechanisms in place. Therefore, the presence of prostaglandins may both promote and block DC-dependent T cell activation depending on the context of exposure. Additionally, while PGE2 has been largely thought to be pro-inflammatory, recent studies have suggested that PGE2 and prostaglandin analogs may be anti-inflammatory in cases of autoimmune diseases such as systemic lupus erythematosus (13
), due to its effects on DC-mediated cytokine production and shifting immune response from a Th1 to Th2 profile. Inhibition of the T cell receptor stop signal would provide an additional anti-inflammatory mechanism for PGE2.
Interactions between dendritic cells and T cells play a central role in the pathogenesis of autoimmune diseases such as SLE and represent an important therapeutic target. In addition to affecting the T cell receptor stop signal, we found that PGE2 significantly impaired T cell-DC interactions and DC-induced T cell proliferation (). PGE2 and certain PG analogs are FDA-approved agents, and the novel effects on T cell stop signal and interactions with dendritic cells suggests they may have therapeutic benefit in patients with SLE. In support of this possibility is a recent paper that suggests that PGE2 also inhibits IFN-alpha secretion by plasmacytoid dendritic cells, key players in SLE pathogenesis (13
). Additionally, another report recently demonstrated that cyclooxygenase (COX) inhibitors disrupt resolution of inflammation that was dependent upon PGE2 in a mouse arthritis model (12
). Moreover, our results may help to explain why exacerbation of SLE-like symptoms have been reported in patients treated with (COX) inhibitors (13
), which function to decrease prostaglandin synthesis.
In summary, we have identified small molecules that modulate the T cell stop signal using a novel image-based high throughput screen. Because the approach is activation-based rather than inhibition-based, there are likely to be fewer off-target hits. We have shown that Src kinase inhibitors potently block the T cell stop signal and impair T cell-DC interactions. Our findings suggest that compounds which function either downstream or independently of ZAP-70 and LAT are also capable of reversing the T cell stop signal. The ability of prostaglandins to block TCR-induced Rap1 activation and antagonize the T cell stop signal is especially intriguing and supports the use of this class of compounds as therapeutic agents that may have benefit in autoimmune disease. Likewise, these results may help explain the surprising pro-inflammatory effects sometimes seen with COX-2 inhibitors. Taken together, the findings suggest that small molecules that reverse the migration stop signal in vitro may either impair proximal TCR signaling, inhibit signaling at the level of Rap1, or directly induce random motility, thereby limiting TCR-induced stopping and DC-induced T cell activation. The present study illustrates that high throughput imaging of primary human cells can effectively be used to identify small molecules that alter migration stopping, allowing for further understanding of the molecular mechanisms that regulate antigen-induced T cell arrest, and offering a new paradigm for drug discovery.