The signaling pathway emanating from TCR and leading to the transcriptional activation of the IL-2 promoter is dependent on the second messenger calcium and the calcium and calmodulin-dependent protein phosphatase calcineurin that has been shown to be a common target for both cyclosporine A and FK506 
. We thus engineered a reporter T cell line by stably integrating a luciferase reporter gene under the control of the minimal human IL-2 proximal promoter into the genome of Jurkat T cells. Upon stimulation with PMA and ionomycin, a 20-fold increase in luciferase activity was observed (data not shown). Using the reporter T cell line, we screened the Johns Hopkins Drug Library at a final concentration of 10 µM for each drug using the IL-2 reporter assay in 96-well format 
. The known immunosuppressive drugs CsA and FK506 were both positive hits, validating the screen. One of the most potent novel hits was identified as clofazimine (), a known anti-mycobacterial drug that has been used for the treatment of leprosy 
Clofazimine inhibits IL-2 production and NFAT activation in Jurkat T cells.
The ability of clofazimine to inhibit TCR-mediated IL-2 production was confirmed using transiently transfected IL-2 luciferase reporter gene in Jurkat T cells and a separately prepared clofazimine stock solution. Clofazimine inhibited PMA/ionomycin-stimulated IL-2 luciferase reporter gene activation with an IC50 of 22 nM (). It also inhibited the activation of endogenous IL-2 promoter in response to PMA and thapsigargin with an IC50 of 1.1 µM (). Importantly, clofazimine inhibited human mixed lymphocyte reaction with an IC50 of 0.9 µM (), similar to its effect on endogenous IL-2 production in Jurkat T cells.
The transcription activation of the IL-2 promoter is dependent on three key transcription factors, NF-AT, NF-κB and AP-1. We thus determined whether clofazimine affected the activation of each of those transcription factors using their respective luciferase reporters. As shown in , while the NFAT luciferase reporter was as sensitive to clofazimine as the IL-2 promoter-driven luciferase reporter gene, the NF-κB luciferase reporter is about 40-fold less sensitive to clofazimine. In contrast, the AP-1 luciferase reporter gene activity was enhanced, rather than inhibited, by higher concentrations of clofazimine (). A similar stimulation of the AP-1 reporter was also observed at higher concentrations of CsA (Fig. S1A
), suggesting that clofazimine may affect the same signaling pathway as CsA.
The selective inhibitory effects of clofazimine on both NFAT and NF-κB over AP-1 suggested that it is likely to affect the activation of their common upstream regulator, calcineurin 
. To assess this possibility, we determined whether clofazimine, like CsA and FK506, affected the dephosphorylation of endogenous NFAT in response to ionomycin treatment. Similar to CsA, clofazimine inhibited ionomycin-induced dephosphorylation of NFATc2 in a dose-dependent manner (). In addition, clofazimine also blocked the ionomycin-induced nuclear translocation of NFAT in Jurkat T cells (Fig. S1B–C
). Together, these results indicated that clofazimine inhibited the activation of calcineurin in vivo
. We next examined the effects of clofazimine on the activity of calcineurin in vitro
. Clofazimine had no effect on the enzymatic activity of calcineurin with either para
-nitrophenylphosphate or immunoprecipitated endogenous NFATc2 as a substrate (Fig. S2A–B
). Nor did it affect the binding of GST-NFATc2 to recombinant calcineurin (Fig. S2C
). Interestingly, when the association between the N-terminal fragment of NFAT and the constitutively active form of calcineurin (CnΔC) was examined in a mammalian two-hybrid assay, clofazimine inhibited the calcium-dependent NFAT-calcineurin interaction in a dose-dependent manner (Fig. S2D
). Thus, clofazimine appeared to act at a step upstream of calcineurin activation in vivo
, raising the possibility that it affected either the release of intracellular calcium or calcium influx through the plasma membrane calcium channels.
We employed live cell imaging to determine the effect of clofazimine on changes in intracellular calcium concentrations in response to thapsigargin treatment. Using the calcium indicator dye Fura-2AM, we were able to observe entry of calcium into Jurkat T cells upon treatment of cells with thapsigargin followed by addition of 2 mM Ca2+
into the extracellular medium (). Pretreatment of Jurkat T cells with known CRAC channel inhibitors econazole or gadolinium abrogated calcium entry as expected (). We noticed that Jurkat T cells exhibited heterogeneity in their response to clofazimine with varying degrees of inhibition at a given concentration of clofazimine. For example, when cells were preincubated with clofazimine for 5 min, the calcium entry of only about a quarter of cells are inhibited to near completion by the drug while that in remaining cells was blocked to a lesser degree (). When the preincubation time was increased to up to 2 h, there was a time-dependent increase in the proportion of cells that became sensitive to clofazimine (Fig. S3A–B
). The inhibitory effect of clofazimine on the extracellular calcium influx suggested that it might affect the CRAC channel. We thus examined the effects of clofazimine in reconstituted CRAC channels using ectopically expressed CRACM1 (Orai1), CRACM2 or CRACM3 subunits co-expressed with STIM1 in HEK 293T cells. But we observed no effects of clofazimine on the reconstituted CRAC current (Fig. S3C
), ruling out the possibility that clofazimine directly interacts and interferes with the known components of the CRAC channel.
Clofazimine interferes with calcium influx in Jurkat T cells.
Given that clofazimine inhibited calcineurin activation in T cells, we next determined whether clofazimine affected the oscillation frequency Ca2+
entry in Jurkat T cells, which has been shown to be critical and selective for sustained activation of calcineurin and NFAT to drive cytokine gene expression 
. Indeed, addition of clofazimine significantly disrupted the oscillation patterns of the store-operated Ca2+
entry induced by a low concentration of thapsigargin 
. It both decreased the amplitude and increased the period of the calcium oscillation (). In contrast to the partial response of calcium influx to clofazimine detected by Fura-2AM (), over 80% of cells exhibited elongation of the oscillation period upon treatment with clofazimine, indicating that this effect is statistically significant ().
The pronounced effects of clofazimine on the oscillation patterns of calcium entry, together with the lack of effect of clofazimine on reconstituted CRAC current, raised the possibility that it may affect other channels, particularly potassium channels, which are known to regulate the driving force for Ca2+
through open CRAC channels. We thus determined the effects of clofazimine on the activity of various known channels expressed in activated T cells. As shown in , clofazimine had a dramatic effect on Kv1.3 current in a time- and dose-dependent manner. It inhibited the Kv1.3 potassium current with an IC50
of 300 nM and a Hill coefficient of 0.75 (), consistent with its potency for the inhibition of both endogenous IL-2 production in Jurkat T cells and the human mixed lymphocyte reaction (). In addition to Jurkat T cells, we also determined the effect of clofazimine on Kv1.3 activity in primary human T cells and a similar inhibitory effect was observed as in Jurkat T cells (). In contrast to Kv1.3, the activity of Ca2+
-activated potassium channels (IKCa1) 
and non-selective cation channels (TRPM4) 
, remained unaffected by clofazimine at up to 10 µM concentration when cells were perfused with intracellular solutions in which Ca2+
was buffered to 1 µM (data not shown).
Clofazimine inhibits Kv1.3 channel.
To further investigate the specificity of clofazimine, we conducted a series of experiments testing the drug against several heterologously expressed potassium channels, including mouse Kv1.3 
. As shown in , clofazimine strongly suppressed mouse Kv1.3 stably expressed in L929 cells with an IC50
of 470 nM and a Hill coefficient of 0.5 (). All other Kv channel species tested (Mouse Kv1.1, rat Kv1.2, human Kv1.5 and mouse Kv3.1 
proved considerably less sensitive to clofazimine, blocking less than 50% of current at a 10 µM concentration (Fig. S4
). This indicates that their IC50
values for clofazimine are above 10 µM. Interestingly, there seems to be some voltage dependence to the effect on Kv channels other than Kv1.3, since the clofazimine block is smaller at 0 mV compared to +80 mV (Fig. S5
). Since most cells do not depolarize beyond 0 mV, at least not for appreciable amounts of time, this represents the more physiologically relevant parameter in regards to the inhibitory effect of clofazimine. Taken together, these data suggest that clofazimine is highly selective for the Kv1.3 channel.
Kv1.3 is known to exhibit a polarized cell surface expression pattern, which can be visualized using polyclonal antibodies in conjunction with Cy5-labeled secondary antibodies 
(). We took advantage of the intrinsic fluorescence of clofazimine, which can be detected using the same filter as for FITC and compared the distribution patterns of clofazimine and Kv1.3. Indeed, clofazimine displayed the same polarized subcellular localization pattern as that of Kv1.3 (), suggesting that clofazimine is likely to be associated with Kv1.3 in vivo
Interaction between clofazimine and Kv1.3 in vivo and in vtro.
Next, we assessed the direct interaction between clofazimine and purified recombinant Kv1.3 in vitro
. His-Kv1.3 was expressed in 293T cells and purified to near homogeneity () as described previously 
. The interaction between purified Kv1.3 protein and clofazimine was assessed by taking advantage of the difference in their mobility in native polyacrylamide gels. Free clofazimine migrated quite slowly in the gel (). Addition of BSA did not affect the gel mobility of clofazimine. Upon mixing with recombinant Kv1.3, however, clofazimine co-migrated with Kv1.3, as judged by the overlap of the Kv1.3 protein band revealed by Western blot and the colored clofazimine band (). The shift in gel mobility of clofazimine by recombinant Kv1.3 strongly suggests that clofazimine forms a complex with recombinant Kv1.3 by directly binding to the protein.
To further assess the physiological relevance of Kv1.3 as a molecular target for clofazimine, we first determined the effect of ectopic overexpression of Kv1.3 on the sensitivity of the IL-2 reporter gene to clofazimine. As shown in , overexpression of Kv1.3 led to a gain in resistance of the IL-2 reporter to clofazimine in a dose-dependent manner. At the highest concentration of Kv1.3 expression plasmid used (2 µg), there was a 35-fold increase in the IC50
value of clofazimine. Next, we downregulated the expression of endogenous Kv1.3 using lentivirus-mediated RNA interference. Of a total of nine RNAi constructs tested, the most effective construct, shKv1.3-4, partially downregulated the protein level of Kv1.3 by ca. 70 % (). A comparison of the dose-response curves of Jurkat cells transduced with shKv1.3-4 lentiviruses and those transduced with viruses carrying a control shRNA against EGFP revealed that knockdown of Kv1.3 increased the sensitivity of the IL-2 luciferase reporter to clofazimine with a nearly 5-fold decrease in the IC50
values (). In contrast, knockdown of Kv1.3 had no effect on the sensitivity of the IL-2 luciferase reporter to CsA (Fig. S6
). The changes in the sensitivity of the IL-2 reporter gene to clofazimine upon overexpression or knockdown of Kv1.3 provide strong support for the notion that Kv1.3 is a specific molecular target of clofazimine.
Kv1.3 has been implicated in T cell activation and has served as a molecular target for developing novel immunosuppressive agents 
. Given that clofazimine is already used in the clinic, albeit for a completely different indication, we wondered whether it is efficacious in animal models of organ transplantation. Initial experiments using mouse skin or heart transplant models revealed no beneficial effects of clofazimine in those models. These negative results are not surprising given that Kv1.3 plays distinct roles in humans and rodents, as it has been shown that Kv1.3 is dispensable in mice due to the up-regulation of other chloride channels 
. Consistent with this notion, we also failed to observe a dose-dependent inhibition of IL-2 production in primary mouse T cells (Fig. S7A
) and murine mixed lymphocyte reaction (Fig. S7B
). Moreover, similar results were obtained for mixed lymphocyte reaction using cells derived from rats, making it difficult to evaluate the in vivo
effects of clofazimine using well-established animal models. To overcome this problem, we turned to a model of reconstituted human T cell-mediated human skin rejection in immunodeficient mice 
. We thus transplanted human foreskin into Pfb-Rag2−/− mice that lack T, B and NK cells. Upon healing of the skin graft for about 7 days, a total of 100 million human peripheral blood lymphocytes from an unrelated donor were adoptively transferred into the same animals. The animals were administered orally either olive oil (control) or clofazimine at 50 mg/kg/day for a total of 10 days (). For the control group, the transplanted foreskin was rejected with a median survival time of 11 days (). For the group treated with clofazimine, the skin survived even beyond the cessation of the drug treatment with a mean survival time of 35 days (), which is comparable to the efficacy for FK506 treatment (data not shown). It is noteworthy that in a parallel experiment using murine skin and total murine T cells, clofazimine had no effect on the survival of murine skin transplant (). Together, these results demonstrated that clofazimine is uniquely effective in inhibiting human T cell-mediated graft rejection with no significant effect on murine T cells.
Clofazimine inhibits human T cell-mediated skin graft rejection in immunodeficient mice.