The development of an immune synapse between T cells and APCs is a key step in the events leading to full T cell activation. When T cells interact with APCs, their F-actin and signaling molecules are enriched in the specialized junction between the T lymphocyte and the APC, which consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Actin polymerization at the immune synapse stabilizes conjugate formation and facilitates T cell activation (54
). Ise et al. (55
) have shown that orally tolerant T cells can form conjugates with APCs, but that they are defective in immunological synapse formation. Their results are consistent with ours in that orally tolerant T cells could not translocate TCR, PKC-θ, or lipid rafts to the T cell/APC contact site. Heissmeyer et al. (34
) also demonstrated that immunological synapse formation by in vitro-anergized T cells induced with ionomycin treatment was unstable. Their observations showed by live cell imaging that the synapses formed normally at early time points after incubation on lipid bilayers, but that at later time points these broke down. Such an analysis remains to be carried out in our adaptive tolerance model.
Integrin-mediated adhesion is essential for the formation of stable contacts between T cells and APCs. RAP1 in T cells is a critical activator of these integrins and plays an essential role in LFA1-mediated interaction with ICAMs on the APCs (17
). Adaptively tolerant T cells showed only modest activation of RAP1 upon TCR stimulation, although the basal activity of RAP1 was increased. The almost normal conjugate formation that we observed may be due to the combination of this modest activation of RAP1 and the increased expression of LFA1 (3 fold) observed on the adaptively tolerant T cells. In other models, VAV1 has been shown to be required for integrin-mediated adhesion of T cells to peptide-loaded APCs (56
). The guanine nucleotide exchange factor (GEF) activity of VAV1 was required for conjugate formation and to a lesser extent for integrin activation (57
). However, whether VAV recruitment into the synapse is required for the integrin-mediated adhesion of the T cells to peptide-loaded APCs was unclear in these studies. Our data would suggest that VAV activation without enrichment into the cSMAC is sufficient for conjugate formation.
Nonetheless, VAV normally does play an important role in immune synapse formation, TCR capping and lipid raft clustering into the immune synapse (56
). Interestingly, tyrosine phosphorylation of VAV was not impaired in adaptively tolerant T cells and in fact was sometimes enhanced following stimulation with Ag-APC or anti-TCR/CD28. In addition, this led to normal activation of RAC1 and CDC42. Because Cbl-b
deficient T cells also show enhanced tyrosine phosphorylation of VAV (59
levels were investigated in adaptively tolerant T cells. However, similar to other models of anergy (60
was found to be upregulated instead of down-regulated (S. Choi and R. H. Schwartz, unpublished observations). VAV1 can be tyrosine phosphorylated by Lck in vitro (44
). Fyn also plays a major role in controlling VAV1 phosphorylation after stimulation through the TCR and CD28 (45
). Our studies show that VAV phosphorylation requires Src family kinase activity in both normal and adaptively tolerant T cells as we could completely inhibit the tyrosine phosphorylation with the SRC-family kinase inhibitor PP2 (Supplemental Fig. 3A
and Supplemental Fig. 4A and B
). We have shown previously that LCK activity is only marginally decreased in adaptively tolerant T cells, and that FYN activity is dramatically increased (6
). This enhanced SRC-family kinase activity might be responsible for the increase in VAV1 phosphorylation that we sometimes observed. In other cells this phosphorylation event has been reported to require phosphotidylinositol 3-kinase (PI3-K)-generated lipid products (44
) in order to recruit VAV to the plasma membrane via its pleckstrin homology domain. Furthermore, VAV tyrosine phosphorylation upon costimulation of the T cell with anti-CD3/CD28 has been reported to be PI3-K dependent (61
). In our hands, however, pretreatment with PI3-K inhibitors did not reduce VAV tyrosine phosphorylation following stimulation of naïve transgenic CD4+
T cells with either anti-TCR/CD4 or anti-CD3/CD28 (Supplemental Fig. 4A
) or with MCC peptide-prepulsed APC (Supplemental Fig. 3A
), even though the downstream effector of PI3-K, AKT, and the downstream effector of AKT, GSK3α/β, were both normally phosphorylated upon anti-CD3/CD28 stimulation, in adaptively tolerant T cells (Supplemental Fig. 4C
). Thus, we think that the major pathway for VAV tyrosine phosphorylation is through SRC-family kinases and that VAV must get to the plasma membrane by some other mechanism.
Despite an intact activation pathway for VAV, RAC1, and CDC42 in adaptively tolerant T cells, it was not sufficient to allow VAV1 to be recruited to the immunological synapse (). What then is missing? Surprisingly, adaptively tolerant T cells showed decreased expression of ITK at both the mRNA and protein levels. ITK plays an important role in actin polymerization and in VAV recruitment to the immune synapse (62
). It also plays a key role in regulating TCR-mediated polarization of integrins and signaling molecules to the site of TCR stimulation, as well as the up-regulation of integrin adhesion (63
). Like our results, T cells from ITK−/−
mice show normal tyrosine phosphorylation of VAV (62
). Similarly, loss of ITK expression by siRNA knock down did not alter the pattern of VAV tyrosine phosphorylation, but instead disrupted the interaction of VAV with SLP76 (53
). SLP76 is present in normal amounts in adaptively tolerant T cells; however, interestingly, GADs levels were significantly diminished along with ITK. GADs is a linker protein required to bring SLP76 to the TCR activation complex in the plasma membrane by binding to tyrosine phosphorylation sites on LAT via an SH2 domain interaction (65
). LAT phosphorylation, however, is greatly impaired in adaptively tolerant T cells (), reducing the number of active binding sites for the limited amount of GADs in the cell to find. This combination of negative effects leads to an impairment in SLP76 recruitment to the TCR activation complex. As a consequence, SLP76 cannot be phosphorylated by ZAP70, and this impairs its ability to recruit the limited amount of ITK in the cell to the TCR activation complex. Without ITK mobilization, PLCγ1 is not optimally phosphorylated, leading to an impairment in PIP2 hydrolysis and a failure to generate adequate amounts of the second messengers IP3 and diacylglycerol (DAG) (66
). In addition, the curtailment of ITK binding limits the recruitment of VAV into the immunological synapse.
As a consequence of these signaling defects, we found that enhancement of actin polymerization at and MTOC polarization toward the T cell/APC contact area were greatly impaired in adaptively tolerant T cells. The actin cytoskeleton is critical for T cell signaling and normal T cell-APC conjugate formation (67
). VAV1 regulates this actin polymerization (68
) and thus its reduced presence in the synapse would impair F-actin accumulation in this part of the cell. MTOC polarization is controlled by ZAP70 kinase activity (52
). Recently, Quann et al. (69
) have shown that this polarization is driven by localized accumulation of DAG. Our previous experiments (6
) showed an impairment in ZAP70 kinase activity and a profound defect in PLCγ1 phosphorylation in adaptively tolerant T cells. This PLCγ1 defect may result in a limited production of DAG, which could cause the impairment of MTOC polarization seen in these cells.
In this paper, we have shown that adaptively tolerant T cells retain the ability to form conjugates with APC, but are defective in translocation of TCR signaling molecules into the contact site. The tolerant T cells showed both increased LFA1 expression levels and increased basal RAP1 activity compared to naïve T cells, even though they could only modestly activate RAP1 upon TCR stimulation. These alterations were adequate for initial conjugate formation. In addition, Ag-induced activation of VAV1 was not impaired, only its accumulation into the immunological synapse. ITK has been previously reported to facilitate this recruitment process and interestingly ITK levels were down-regulated in adaptively tolerant T cells. Also, ITK is normally recruited to the TCR-signaling complex by SLP76 following its phosphorylation by activated ZAP70. SLP76 is brought to the complex by GADs, which recognizes ZAP70-phosphorylated tyrosines on LAT. In adaptively tolerant T cells we found that SLP76 levels were normal, but that SLP76 phosphorylation and GADs levels were down-regulated. Thus, defective ITK and GADs expression, in addition to impaired tyrosine phosphorylation of LAT by the ZAP70 kinase, appear to be the major impediments to VAV1 recruitment for enabling formation of a stable immunological synapse.