CypA is a member of the immunophilins that catalyzes the reversible cis-trans conversion of peptide bonds containing the amino acid proline. The two major groups of mammalian immunophilins, the cyclophilins and FK506-binding proteins (FKBPs), function as chaperons and assist newly synthesized proteins to undergo proper folding and acquire a conformation that is essential for their stability, localization, and biological function. The T lymphocyte immunophilins, CypA and FK506, are of particular interest because of their ability to bind the CsA and FK506 (tacrolimus) compounds, respectively, and promote immunosuppression by inhibition of T cell activation [53, 55]. Triggering of the TCR stimulates a rapid phospholipase C (PLC)-mediated breakdown of inositol phospholipids, resulting in the production of second messengers, including inositol trisphosphate (IP3), which promotes the rise in intracellular free Ca2+ concentration and the activation of calcineurin . Once activated, the calcineurin associates with and dephosphorylate the nuclear factor of activated T cells (NFAT) , which can then translocate to the nucleus, bind to selected DNA promoter regions, and initiate the transcription of interleukin-2 (IL-2)  and other proinflammatory cytokines .
Inhibition of calcineurin by immunophilin-drug complexes [60, 61] hinders NFAT translocation to the nucleus and inhibits the formation of IL-2 and other cytokines which are crucial for the maintenance, survival, differentiation, and activation of distinct T cell subtypes . While inhibition of calcineurin activity is a major mechanism by which immunophilin-drug complexes induce immunosuppression, immunophilins were found to impair T cell functions by intervening with the activity of several additional effector molecules. Functional studies demonstrated that immunophilin-drug complexes block the activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) in TCR-stimulated T cells, via a calcineurin-independent mechanism . p38 is a direct substrate for activated ZAP70 in TCR-engaged T cells, and phosphorylation of p38 initiates a negative feedback loop that promotes the dissociation of ZAP70 from CD3ζ and negatively regulates TCR proximal signals . Based on the above data, the effect of immunophilin-drug complexes on p38 may be indirect, reflecting the effect of the immunophilin-drug complexes on ZAP70, which is an upstream regulator of p38.
Studies by Brazin et al.,  revealed that CypA can form a stable complex with Itk, an essential kinase for signal transduction downstream of activated TCR, which plays a key role in T cell activation, proliferation, and differentiation [65–67]. CypA binding to Itk induces a proline-dependent conformational switch within the Itk SH2 domain leading to inhibition of Itk enzymatic activity and modulation of the Itk ligand recognition, a mechanism that can be disrupted by the presence CsA . Further studies revealed that irrespective of CsA, CypA can downregulate TCR signal strength in CD4+ T cells . CT10 regulator of kinase II (CrkII) adaptor protein is an additional TCR-coupled signaling protein that is regulated by CypA. In vitro studies of a recombinant protein consisting of the SH3N-linker-SH3C of the chicken CrkII showed that this peptide can undergo CypA-mediated cis-trans isomerization at the linker region Pro238 residue [69, 70]. The cis conformer of CrkII is autoinhibited due to the intramolecular interaction between its two SH3 domains. In contrast, the trans conformer of CrkII acquires an extended conformation in which its SH2 and SH3 domains are available for interaction with binding partners. TCR stimulation promotes a direct physical interaction between CrkII and ZAP70, which is mediated by the Crk-SH2 domain and phospho-Tyr315 in the ZAP70, an interaction that is dependent on the presence of an active Lck [27, 30]. Both CypA and FKBP were found to associate with CrkII in resting Jurkat T cells and regulate its conformation . In addition, CypA increased the ability of CrkII to interact with the guanine-nucleotide releasing factor (C3G), which promotes integrin-mediated cell adhesion and migration . As a result, CsA/FK506-mediated inhibition of PPIase decreased the ability of T cells to adhere to fibronectin-coated surfaces and migrate toward the stromal cell-derived factor 1α, suggesting that CsA/FK506 interferes with the PPIase-mediated, CrkII-dependent mechanisms that regulate selected effector T cell functions .
In the present study, we found that CypA association with ZAP70 peaks at about 1 min post TCR stimulation, when Lck-mediated phosphorylation of ZAP70 is near maximum, suggesting that CypA regulates predominantly the phosphorylated and not the non-phosphorylated ZAP70. The results also suggest that CypA-mediated regulation of ZAP70 in vivo occurs in TCR-stimulated, and not in resting T cells, and that CsA prevents the TCR engagement-dependent formation of CypA-ZAP70 complexes. The early event which promotes ZAP70 association with activated TCR is phosphorylation of the CD3 chain ITAMs, predominantly those of the CD3ζ. We found that CypA association with ZAP70 enables its recruitment to the vicinity of the activated TCR within the IS. In agreement, the time kinetic of CypA-ZAP70 association paralleled that of the ZAP70 recruitment to the IS of TCR-stimulated T cells. Furthermore, CsA was found to inhibit CypA association with ZAP70 and prevent the colocalization of CypA with ZAP70 at the cell membrane. The results suggest that catalytically active CypA plays a role in the conformational regulation of ZAP70, perhaps by isomerization of ZAP70, although conformational constraints imposed on ZAP70 due to its association with CypA might also affect ZAP70 activity by modulating its ability to undergo phosphorylation and/or interaction with binding partners and substrate proteins.
The finding showing that cell treatment with CsA augment the phosphorylation of LAT, an immediate substrate of ZAP70 in TCR-stimulated T cells, is a reminiscent of the observation made in studies of the regulation of another CypA-interacting T cell PTK, the Itk . In both cases, inclusion of CsA at a time point in which CypA binds its target PTK (either Itk or ZAP70) augments the PTK-induced phosphorylation of its respective primary substrates (PLCγ1 and LAT, respectively). These observations highlight the notion that CypA functions as a negative regulator of PTKs in the early phases of T cell activation.
Studies in Jurkat T cells that express the ZAP70 activity-biosensor, ROZA-XL, revealed that CsA increases ZAP70 activity at 1 min post TCR-stimulation of T cells, indirectly implying that CypA is a negative regulator of the ZAP70 catalytic activity and that CsA can reverse the effect of CypA on ZAP70. This in vivo assay is based on the ability of active ZAP70 to phosphorylate a Tyr-containing LAT epitope in the biosensor protein, an epitope that its phosphorylation directly correlates with ZAP70 activity. Recent studies demonstrated that Lck-mediated phosphorylation of ZAP70 is a crucial step for ZAP70 bridging to LAT  and the present studies further demonstrates that ZAP70 phosphorylation by Lck is required for the ZAP70 interaction with CypA.
Notably, inclusion of CsA in the assay system at a late time point of cell activation (10 min), when CypA association with ZAP70 is negligible, CsA had no effect on ZAP70-mediated substrate phosphorylation. Complexes of CsA-CypA are known to interact with and inhibit the cytoplasmic phosphatase, calcineurin, and thereby its primary target, the NFAT transcription factors [61, 73]. More recent studies reported the recruitment of calcineurin to the TCR signaling complex, where it reverses inhibitory phosphorylation on Lck and indirectly promote the activation of ZAP70 . It is possible therefore that some of the in vivo effects of CsA on ZAP70 that were observed in the present studies reflect the CsA-CypA-mediated inhibition of calcineurin, which prevent the activation of Lck, and its downstream protein, ZAP70. In vitro kinase assay of ZAP70 performed in the absence or presence of enzymatically active human recombinant CypA demonstrated that CypA induces a time- and concentration-dependent reduction in the extent of ZAP70 autophosphorylation as well as substrate phosphorylation (cfb3). Inclusion of CsA in the preincubation step reversed the effect of CypA on ZAP70. These results suggest that CypA, which associates with ZAP70 in TCR-engaged T cells, can impose its effect on ZAP70 activity via a direct mechanism. We hypothesize that CypA modulates ZAP70 activity by isomerization of ZAP70 and/or physical interaction with a ZAP70 motif that alters the catalytic activity of ZAP70 and/or accessibility to substrates or ATP. The observation that Pin1, in contrast to CypA, does not affect ZAP70 catalytic activity suggests a selectivity in PPIases towards ZAP70.
Our data support the hypothesis that maximal phosphorylation of ZAP70 at ~60 sec post TCR stimulation involves the concomitant association of ZAP70 with CypA which can modulate the catalytic activity of ZAP70 (Fig. 6). Under the assays conditions we have used, CypA was found to inhibit ZAP70 activity. However, CypA can interconvert both cis and trans isomers and the preferred direction of ZAP70 isomerization under physiological conditions might be determined by the phosphorylation status of ZAP70 or its association with selected binding proteins. The results suggest that CypA functions as a physiological regulator of ZAP70 that might contribute to the amplitude, duration, and fine-tuning of the T cell activation response.