- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data. © 2013 International Society for Advancement of Cytometry
The short-term activation of T lymphocytes, especially that of autoreactive T cells, plays a key role in initiating and maintaining most autoimmune reactions, including that observed in rheumatoid arthritis (RA). The transient increase of the cytoplasmic free calcium level ([Ca2+]cyt) in T lymphocytes is an indispensable part of this process. The engagement of the TCR/CD3 complex upon antigen presentation leads to the activation of several transmembrane signaling pathways which result in Ca2+ release from intracellular stores. This is followed by further Ca2+ entry from the extracellular space through the store-operated calcium release activated calcium (CRAC) channels. Voltage-gated Kv1.3 and calcium-dependent IKCa1 K+ channels play a pivotal role in the maintenance of [Ca2+]cyt, since they sustain the electrochemical potential gradient needed for further Ca2+ entry by the efflux of K+ from the cytoplasm (1, 2). Specific inhibition of these channels results in diminished Ca2+ influx and a lower level of lymphocyte activation and proliferation (3, 4).
This observation offers a unique opportunity for targeted pharmacological intervention with improved specificity from existing drugs against disease-causing autoreactive lymphocytes. According to Beeton et al., disease-associated autoreactive T cells are mainly CCR7− CD45RA− effector memory T cells (TEM cells) with elevated Kv1.3 channel expression, in comparison with naive and central memory T cells (TCM cells) that express low levels of Kv1.3 channels. Consequently, Kv1.3 inhibitors primarily suppress the activation and proliferation of the autoantigen-specific TEM cells, while sparing other classes of T cells (5). Beeton et al. also evaluated the therapeutic effects of Kv1.3 inhibitors in a rat model of RA. The animals treated with the specific Kv1.3 channel blockers showed significantly less joint deviations and significant improvement in radiological and histopathological findings (6). In addition, no clinical side-effects or signs of toxicity were identified during the trial. However, our earlier trial on human samples from patients with RA showed that Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells. Thus the inhibition of Kv1.3 channels does not seem to be specific enough in peripheral human RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent (7).
Flow cytometry is a suitable method for the sequential determination of [Ca2+]cyt in millions of stimulated lymphocytes over a time period. It also enables the comparison of Ca2+ influx kinetics in more than one lymphocyte subsets of the same sample simultaneously. However, until recently, there had been an unmet need for a method that can reliably compare kinetic flow cytometry data. A number of approaches have been available as part of various flow cytometry software for characterizing kinetic measurements. These approaches calculate median fluorescence of the dye of interest and apply a smoothing method in order to describe given parameters of the recording, such as the area under the curve (AUC) value (8).
Our research group developed a robust algorithm (FacsKin) that fits functions to median values of the data of interest and calculates relevant parameters describing each function. By selecting the best fitting function, this approach provides an opportunity for the mathematical analysis and statistical comparison of kinetic flow cytometry measurements of distinct samples (9).
In this study, we used flow cytometry to characterize Ca2+ influx kinetics upon activation in major T cell subsets (CD4, Th1, Th2, CD8) isolated from patients with recently diagnosed and established RA and their sensitivity to the specific inhibition of Kv1.3 and IKCa1 lymphocyte K+ channels. For the evaluation of kinetic flow cytometry data, we utilized FacsKin, our novel algorithm (9), and the kinetic module of FlowJo, a currently available flow cytometry software. We hypothesized that FacsKin enables the discovery of more differences between Ca2+ influx kinetics of the investigated RA groups and provides a more precise characterization of kinetic measurements than currently available methods.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
- Supporting Information
In our investigation, we aimed to compare peripheral T lymphocyte Ca2+ influx kinetics upon activation in two different subgroups of RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms.
After analyzing data with FacsKin, significant differences could be observed in Ca2+ influx kinetics between patients with recently diagnosed RA and patients with established RA (Supporting Information Table 1). Our results revealed that the tmax value of the Th2 subset in patients with established RA is reached slower compared to patients with recently diagnosed RA, indicating that Th2 cells respond slower to activating stimuli as the disease duration increases. Th2 cells mainly regulate anti-inflammatory responses, thus this finding possibly indicates the remission of immunoregulatory process along with the duration of the disease. This is in contrast with our earlier findings in RA, which presented an elevated Th2 response in the early stages of RA compared to healthy controls (Supporting Information Table 1) (7), presumably to control the ongoing inflammation. On the contrary, we measured lower tmax values in the CD8 subset of patients with established RA, which may indicate an enhanced direct cytotoxic response, raising the notion that the increasing duration of the disease results in an even greater imbalance between the pro- and anti-inflammatory responses. The peak of calcium influx in lymphocytes isolated from patients with RA is reached more rapidly in the CD4+ subsets compared to healthy individuals, indicating that they respond more quickly to stimulation, probably due to the ongoing autoimmune response.
Upon treatment with specific inhibitors of the Kv1.3 and IKCa1 channels (MGTX and TRAM, respectively), AUC values decreased in all subsets in both RA study groups. However, the sensitivity of the two study groups was different to lymphocyte K+ channel inhibition. Blocking the Kv1.3 channel decreased Ca2+ influx in the CD4, Th2, and CD8 subsets of patients with established RA to a lower extent than in patients with recently diagnosed RA. However, Th1 cells were more sensitive to the inhibition of Kv1.3 channels in patients with established RA. In patients with recently diagnosed RA, Max values decreased in all subsets upon treatment with MGTX, while in patients with established RA, the Max value decreased in the Th1 subset only. Similarly, upon the inhibition of the IKCa1 channel, Th1 cells of patients with established RA showed a higher level of decrease of the AUC and Max values than those of patients with recently diagnosed RA.
The above results could either be due to the altered expression or different functionality of Kv1.3 channels. Therefore, we measured the cell surface expression of these channels on the investigated lymphocyte subsets in a semi-quantitative manner using a specific antibody. However, we could not detect a difference in the expression of Kv1.3 channels between the two study groups. Hence, we presume that functional alterations are responsible for the altered sensitivity to Kv1.3 channel inhibition in patients with established RA. Of note, due to the lack of commercially available antibodies against IKCa1, we could not investigate its expression in a similar manner.
Our findings indicate that while initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. Based on our results, it is difficult to tell whether this difference is due to the primary course of the disease or whether it is influenced by pharmacological treatment.
We also compared the AUC values, the most representative parameter for kinetic measurements, using the kinetic module of FlowJo (Supporting Information Table 2). However, the above difference between the sensitivity of recently diagnosed and established RA lymphocytes to K+ channel inhibition could not be demonstrated based on results obtained with this approach. In fact, we could not observe any effect of the applied inhibitors in the established RA group when data were analyzed with FlowJo. A plausible explanation for this might be that FlowJo is based on a smoothing method of median fluorescence values performed by moving average calculation. However, due to the non-normal distribution of flow cytometry data (13), techniques that assume normality (such as those calculating average or standard deviation) lead to potentially incorrect smoothing and artifacts in the kinetic parameter (8).
With our method of function fitting and selecting the best fitting function for the kinetics of the studied biological mechanism instead of smoothing, more statistically significant differences of K+ channel inhibition between the recently diagnosed and established RA groups could be demonstrated. The use of our algorithm largely improves the evaluation of kinetic flow cytometry compared to the current, non-standardized approaches that largely depend on the observers' personal experience, revealing more detailed information and delicate differences of the investigated kinetic process (8, 9).
A limitation of our study is the use of cell surface chemokine receptors instead of intracellular cytokine staining for the identification of Th1 and Th2 cells. The option to use intracellular cytokines as markers of the T helper subsets in our current experiment had to be excluded, since permeabilization of the cell membrane to stain intracellular cytokines would have prevented us from studying calcium influx. However, literary data and our experience indicate that the applied cell surface chemokine receptor markers are sufficient for the identification of the investigated T helper subsets (14, 15).
In conclusion, following evaluation with FacsKin, we were able to describe numerous differences in Ca2+ influx kinetics of major peripheral blood T lymphocyte subsets of patients with recently diagnosed RA and patients with established RA which could not be detected using currently available approaches employing smoothing methods. We found that Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. These data demonstrate that the function fitting algorithm of FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.