t lymphocytes are of paramount importance in many intercellular reactions , such as the regulation of the inflammatory response and immune reactivity . upon the engagement of the t cell receptor ( tcr ) , a number of signal transduction pathways culminate in the transient elevation of the cytoplasmic calcium concentration ( [ ca2+]cyt ) . first , ca2 + is released from intracellular stores that is followed by further ca2 + entry from the extracellular space through the store - operated calcium release activated calcium ( crac ) channels . in the course of lymphocyte activation , k+ channels maintain the driving force for sustained ca2 + influx as they grant the efflux of k+ from the cytoplasm , thus conserving an electrochemical potential gradient between the intra- and extracellular spaces . there are two major types of k+ channels in t cells : the voltage - gated kv1.3 and the ca2 + -activated ikca1 channels . the relation between the ca2 + currents through crac channels and the efflux of k+ makes the proliferation and activation of lymphocytes sensitive to pharmacological modulation of kv1.3 and ikca1 channels , and provides an opportunity for targeted intervention . specific inhibition of these channels results in a diminished ca2 + influx in lymphocytes and a lower level of lymphocyte activation . previous data suggest that selective modulation of lymphocyte activation through specific inhibition of k+ channels may be a possible therapeutic approach for the treatment of autoimmune disease [ 1 - 6 ] . showed that terminally differentiated effector memory t ( tem ) cells play a pivotal role in the pathogenesis of autoimmunity . for instance , wulff et al . suggest that disease causing tem cells are able to home to inflamed tissues in the cns and exhibit immediate effector function in autoimmune disease . they described that the characteristic k+ channel phenotype of tem cells in ms is kv1.3high ikca1low , contrasting naive and central memory t ( tcm ) cells , which exhibit a kv1.3low ikca1high channel phenotype . therefore the therapeutic relevance of specific kv1.3 channel inhibitors is of outstanding interest , as they may offer the possibility for selective modulation of pathogenic tem cells , while naive and tcm cells ( needed for physiological immune responses ) would escape the inhibition through upregulation of ikca1 channel expression . demonstrated that the symptoms of experimental autoimmune encephalitis , a murine model of ms , significantly improved after treatment with selective kv1.3 inhibitors [ 1 , 4 ] . besides naive and memory cells , helper ( cd4 ) and effector ( cd8 ) t lymphocytes modulate the autoimmune response in different ways . cd4 cells influence the immune system by producing cytokines , while cd8 cells are capable of causing immediate cell destruction . th1 cells mainly produce pro - inflammatory cytokines , thus sustaining autoimmune reactions . however , th2 cells reduce the inflammatory response by producing anti - inflammatory cytokines . until the recent past , single - cell techniques were used for the investigation of ca2 + influx during lymphocyte activation . there has been no high - throughput method available to study the kinetics of lymphocyte activation in more subsets simultaneously . single - cell techniques are restricted by not being capable of characterizing this process in complex cellular systems , therefore ignoring the interaction between the different lymphocyte subsets that may modulate the course of their activation . therefore , over the recent years we have created a novel approach that allows simultaneous recording of ca2 + influx in several lymphocyte subsets . 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 . for example , in case of ca2 + influx measurements , the software fits a double - logistic function on each recording . this function is used to describe measurements that have an increasing and a decreasing intensity as time passes . the software also calculates parameter values describing each function , such as the maximum value , the time to reach maximum value or the area under the curve ( auc ) . these parameters represent different characteristics of lymphocyte ca2 + influx kinetics ( figure 1 ) . briefly , peripheral blood mononuclear cells ( pbmcs ) were separated from freshly drawn peripheral venous blood of the investigated subjects . cell fluorescence data were measured and recorded for 10 minutes in a kinetic manner on flow cytometer . our studies were adhered to the tenets of the most recent revision of the declaration of helsinki . over the recent years , we have investigated ca2 + influx characteristics in a number of autoimmune disorders and under different physiological conditions . in this review , we aim to give a brief summary of our findings and of the common characteristics of ca2 + influx in autoimmune disease . they regulate the ongoing inflammatory process of the central nervous system ( cns ) which leads to the damage of the myelin sheath and axons . however , only a small part of t lymphocytes are myelin - specific autoreactive cells . besides the demyelinating action of these cells of the cns , the activation of peripheral lymphocytes also contributes to the pathogenesis and disease progression . in our investigations we measured samples of healthy individuals and ms patients with no immunomodulatory therapy , as well as ms patients treated with interferon beta ( ifn - b ) , currently regarded as the most effective therapy of ms . in our study we discovered therefore , from the cd4-cd8 point of view , we demonstrated specific immunomodulation that may be beneficial in the therapy of ms via the selective suppression of cd8 effector lymphocytes over cd4 helper cells . however , this specificity is not present within the cd4 subset , since our results suggest that th1 and th2 cells are similarly suppressed upon the inhibition of kv1.3 channels . since the cytokine balance is of utmost importance in the regulation of the autoimmune reaction , the inhibition of the th2 subset would probably result in a setback of therapeutic efforts in ms . our findings are relevant in the light of observations regarding the contribution of tem cells to the development of ms , as described above . although the kv1.3high ikca1low pattern is found in both cd4 + and cd8 + tem cells , we can assume that the majority of tem cells are cd8 + , since tem cells express immediate effector function . this provides an explanation for the increased sensitivity of cd8 cells to kv1.3 channel inhibition in ms in our study . we have also demonstrated important differences in ca2 + influx kinetics and lymphocyte k+ channel function in ms patients without ifn - b compared with healthy individuals . the selective blocker of the lymphocytes kv1.3 channel might be a promising drug in combination therapy , supplementing the presently used ifn - b treatment . our results indicate that ifn - b therapy causes compensatory changes only in the th1 subset in ms regarding ca2 + influx kinetics and the function of k+ channels . however , the increased function of the th2 subset , and therefore the production of anti - inflammatory cytokines is less affected . this might contribute to a more effective treatment of the autoimmune process in this disorder . the short - term activation of peripheral blood and synovial fluid t lymphocytes , especially that of autoreactive t cells plays a crucial role in initiating and maintaining the chronic inflammation in the joints of patients suffering from rheumatoid arthritis ( ra ) . these cells regulate the inflammatory process resulting in the destruction of the articular cartilage and also play a role in extra - articular damage . we investigated t lymphocyte ca2 + influx kinetics following activation in peripheral blood of recently diagnosed ra patients compared to healthy individuals . our results indicate that margatoxin ( mgtx ) , a specific blocker of the kv1.3 channel acts differentially on ca2 + influx kinetics in major peripheral blood lymphocyte subsets of ra patients : th2 and cd8 cells are inhibited more dominantly than th1 and cd4 cells . kv1.3 channel expression in ra patients and healthy subjects based on our results , the immunomodulatory effect of kv1.3 channel inhibition is predominantly seen in cytotoxic ( cd8 ) t cells in ra . however , this effect does not seem to be as specific as reported before by beeton and colleagues in case of tem cells , since anti - inflammatory th2 cells are also affected to a noteworthy extent . this subset has an important role in counterbalancing the peripheral lymphocytes might be the differential distribution of disease - associated autoreactive t cells in ra patients on local and systemic levels . in the synovial fluid ( locally ) , autoreactive tem cells , however , this kv1.3 pattern was not detected in peripheral blood t cells , because autoreactive tem cells are infrequent in the circulation . a reason for limited specificity of kv1.3 inhibition in peripheral lymphocytes might be the differential distribution of disease - associated autoreactive t cells in ra patients in the synovial fluid and the circulation . type 1 diabetes mellitus ( t1 dm ) is an autoimmune disease affecting the pancreas by destroying the insulin - producing beta cells . due to the lack of insulin glucose levels both kv1.3 and ikca1 channels contribute to the maintenance of ca2 + influx upon activation . on the contrary , the sensitivity of t1 dm lymphocytes to the inhibition of kv1.3 channels is increased . our data indicate that by specific inhibition of kv1.3 channels , lymphocyte activation can be modulated in t1 dm . it has been hypothesized that beneficial effects of kv1.3 channel inhibition by mgtx are due to the dominance of kv1.3 channels on activated tem cells . it was also presumed that mgtx would inhibit the activation of cd8 lymphocytes responsible for cytotoxic destruction of pancreatic beta cells . nevertheless , our findings obtained with a functional method ( i.e. kinetic flow cytometry measurements ) provide clear evidence for kv1.3 channels to have an important role in each lymphocyte subset in t1 dm , including th2 lymphocytes acting as counterbalancing factors in the development of t1 dm through the production of antiinflammatory cytokines . therefore , administration of kv1.3 channel inhibitors would not have an exclusive effect on cells responsible for the autoimmune response in t1 dm , but may have an impact on the activation characteristics of immune cells in general , leading to unpredictable alterations . for this reason , further studies are needed to describe the effects of the application of specific kv1.3 inhibition and the extent of beneficial consequences in t1 dm . ankylosing spondylitis ( as ) is a chronic inflammatory rheumatic disease , the best characterized of the diseases belonging to the concept of spondylarthritides . the pathogenesis of as is still unclear , but it is considered to be a systemic autoimmune disease . this is supported by the number of alterations found in lymphocyte subgroups in peripheral blood . specifically , increased numbers of circulating th2 helper lymphocytes   were reported in as . along with the alterations observed in cell prevalence one can assume that t cell activation properties may also be altered in as . we investigated the short - term t cell activation characteristics in as before and during infliximab ( ifx ) therapy . cd4 and cd8 cells presented with a delayed increase in cytoplasmic ca2 + levels after activation in as compared to controls . with ifx , therapy the delay in cd4 + cytoplasmic ca2 + levels did not normalize in as . for cd8 + cells , cytoplasmic and mitochondrial ca2 + kinetics during activation normalized by week 6 on ifx ( but not on week 2 ) . of note , the increase of cytoplasmic ca2 + levels in cd4 and cd8cells from as is delayed compared to controls . the delayed ca2 + response of as fits well into the observations done in in vitro tests with t cells exposed to tnf-. church et al . reported that tnf- suppressed the ca2 + peak after pha stimulation ; they have suggested that either signalling pathways upstream of ca2 + mobilisation or the ca2 + signalling itself were impaired by prolonged tnf- exposure . in as , only one study has been performed with a more robust analytical approach : lee et al . did not observe a significant difference in intracellular ca2 + between as patients and normal controls in activated peripheral blood mononuclear cells . the inconsistency between their and our results is probably due to different methodology and cell types investigated . in healthy pregnancy ( hp ) , the maternal immune system needs to acquire tolerance to protect the developing fetus from harmful immunological reactions . if this tolerance is impaired , a general maternal immune response may arise , resulting in systemic inflammation . this has been suggested to be a major factor in the pathogenesis of preeclampsia ( pe ) [ 20 , 21 ] . this disorder is characterized by hypertension , proteinuria , edema and endothelial dysfunction usually evolving in the third trimester of pregnancy . we compared the activation - elicited ca2 + influx in major peripheral t lymphocyte subsets of hp and pe women to that in non pregnant women and tested the alteration of ca2 + influx upon treatment with specific inhibitors of the kv1.3 and ikca1 k+ channels . our findings suggest that the ca2 + influx kinetics in activated t lymphocytes markedly differs in hp compared to non pregnant women : the decreased activation of the th1 subset may partly be responsible for the well - established th2/th1 skewness in hp [ 22 , 24 ] . indeed , the maintained activation properties of th1 lymphocytes in patients with pe may contribute to the lack of th2 dominance associated with normal pregnancy . thus , the decrease in their cytotoxic activity characteristic for hp is not present in pe . it is of particular interest that ca2 + influx of th2 lymphocytes in hp was insensitive to k+ channel inhibition , while ca2 + influx decreased significantly in non pregnant upon treatment with specific channel blockers . of note , th2 lymphocytes in pe presented with non pregnant - like characteristics and were sensitive to k+ channel inhibition as well . while it is unclear whether the resistance or sensitivity of th2 lymphocytes to k+ channel inhibition is reflected in th2 function , it is tempting to speculate that this may be an element contributing to the th2 shift present in hp , but absent in pe . this hypothesis may be supported by reports suggesting that the shape of ca2 + influx influenced by k+ channel functions may determine the cytokine production profile of helper t lymphocytes . interestingly , other differences were also observed between hp and pe . while ca2 + influx in cd8 and th1 lymphocytes was resistant to k+ channel inhibition in pe , that of hp lymphocytes was sensitive . again , while it is unclear whether the resistance of th1 lymphocytes to k+ channel inhibition is reflected in their function , the insensitivity of the th1 subset in pe may be linked to the th1 skewness . comparing the inhibitory properties of lymphocyte k+ channels in non pregnant women and pe our results indicate marked differences of ca2 + influx kinetics and sensitivity to lymphocyte k+ channel inhibition in major lymphocyte subsets between non pregnant , hp and pe lymphocytes . it is of interest that these properties in pe are more comparable to non pregnant than to hp . these results suggest that there is a characteristic pattern for ca2 + influx and its sensitivity to k+ channel inhibition in hp that is missing in pe . this raises the notion that lymphocyte ca2 + handling upon activation may have a role in the characteristic immune status of healthy pregnancy . reduced functioning is well characterized by a lower level of cytokine production compared with adult t cells [ 25 , 26 ] . several factors might be responsible for the decreased cytokine expression compared with that of adult lymphocytes . among many others , the majority of these cells is naive ( cd45ra ) lymphocytes in contrast to adults , where effector ( cd45ro ) cells dominate . another possible factor might be the impairment of mechanisms regulating short - term activation of lymphocytes compared with adults . kv1.3 and ikca1 lymphocyte k+ channels are essential components of these mechanisms . we hypothesized that short - term t lymphocyte activation properties are different in neonates compared with adults . the aim of our study was to characterize the ca2 + influx kinetics upon activation in major t lymphocyte subsets in the neonate and its sensitivity to the specific inhibition of kv1.3 and ikca1 lymphocyte k+ channels , important regulators of ca2 + influx . ca2 + influx following pha activation of t lymphocytes markedly differs in the neonate from tha in the adult . upon treatment of lymphocytes with selective inhibitors of the kv1.3 and ikca1 channels ( mgtx and tram , respectively ) , ca2 + influx reduction was not demonstrated in neonatal lymphocytes only in cd8 subsets . the finding that neonatal lymphocytes are less sensitive to the specific inhibition of k+ channels compared with adults may be due to altered functionality or a lower expression of these channels . therefore , we measured the expression of kv1.3 k+ channels on the investigated lymphocytes . instead of lower values , we found increased expression of kv1.3 channels on neonatal cd4 , cd8 and th2 lymphocytes compared with adults . thus , the option that the decreased sensitivity of lymphocytes to k+ channel inhibition is due to lower channel expression should be excluded . based on the lower sensitivity of lymphocyte ca2 + influx to inhibition at higher channel expression values , it is reasonable to postulate that neonatal kv1.3 channels are functionally altered . signs of altered sensitivity to k+ channel inhibition were present in all major lymphocyte subsets investigated ( i.e. th1 , th2 , cd4 and cd8 cells ) . the only subset in which the short - term activation was inhibited significantly by specific blockers of both kv1.3 and ikca1 channels was cd8 lymphocytes . however , even in this case , the level of inhibition did not reach the extent described in adults in spite of high kv1.3 expression values . furthermore , an interesting observation is that the decreased ca2 + influx found in the cd8 subset in neonates after mgtx and tram treatment is coupled with a massive elevation in the expression of kv1.3 channels by this subset . however , no correlation was detected between ca2 + influx parameters and channel expression data ; thus , a causative relation between the two findings is unlikely . our results may partly explain why neonatal lymphocytes are less responsive to activating stimuli and , hence , exert a lower intensity of immune response . we demonstrated that short - term activation and associated intracellular ca2 + influx kinetics are lower in neonatal lymphocytes compared with adults . our results improve the understanding of the mechanisms that prevent neonatal t lymphocytes from adequate activation upon activating stimuli . they show that the functional impairment of lymphocyte k+ channels may be of importance in those mechanisms . based on our results , a number of dominant features were identified that were present in most of the investigated autoimmune diseases . first , the time when the peak of ca2 + influx was reached decreased in autoimmune patients compared to healthy individuals , indicating that these cells are in a state of sustained reactivity due to the ongoing autoimmune reaction . earlier studies were limited to the investigation of k+ channels in naive and memory lymphocytes . as an extension of these findings to other significant t lymphocyte subsets , in th1 cells in patients suffering from an autoimmune disease we detected a different pattern of sensitivity to the inhibition of lymphocyte k+ channels compared to healthy individuals : a greater decrease of ca2 + influx upon the inhibition of the kv1.3 channel than that of the ikca1 channel was observed . on the contrary , in healthy individuals this finding is of special interest , since th1 cells are regarded as key players in the mediation of pro - infalmmatory responses . however , the selectivity of the investigated inhibitors ( mgtx and tram ) was limited in our experiments , as they did not only affect a single subset , as previously suggested . although in earlier observations the inhibition of kv1.3 channels specifically blocked the function of tem cells , our investigations extending to other significant t lymphocyte subsets demonstrated that the inhibitory effect is present not only in disease - associated cd8 and th1 cells , but also in the anti - inflammatory th2 subset . the induced decrease in their function could lead to unexpected potential side - effects in vivo and also in a setback of therapy , since th2 cells are responsible for anti - inflammatory responses . the advantage of our newly developed method compared with the single - cell techniques is that it enables characterizing the progress of lymhocyte activation in complex cellular systems , since the interaction between the different cell subsets is also taken into account . substances produced during lymphocyte activation ( e.g. cytokines , chemokines ) modulate the function of other cell subsets . thus cell activation does not only affect one cell type but indirectly takes an effect on other cells in the sample . accordingly , different lymphocyte subsets might be regarded as a network , where cells are in connection with the ones surrounding them . furthermore , not only k+ channel blockers , but other molecules and drugs might be screened . investigating other lymphocyte subsets of interest is also possible with our method depending on the type of surface markers used for discriminating the cell types . furthermore , our method may play a key role in several other fields of basic and clinical research where the characterization of different intracellular kinetic processes are needed that can be identified with fluorescent reagents ( e.g. the generation of reactive oxygen radicals , alterations of membrane potential , etc ) .
t lymphocytes are of paramount importance in many intercellular reactions , such as the regulation of the inflammatory response and immune reactivity . until the recent past 
 , single - cell techniques were used for the investigation of calcium influx during t lymphocyte activation . 
 therefore , over the recent years we have created a novel approach that allows simultaneous recording of calcium influx in several lymphocyte subsets using flow cytometry . 
 our research group developed a robust algorithm ( facskin ) for the evaluation of the acquired data that fits functions to median values of the fluorescent marker of interest and calculates relevant parameters describing each function.over the recent years , we have investigated calcium influx characteristics applying this method in a number of autoimmune disorders and under different physiological conditions ( such as the neonatal period and pregnancy ) . in this review , 
 we aim to give a brief summary of our findings and of the common characteristics of calcium influx in the investigated disorders , namely : multiple sclerosis ( ms ) , rheumatoid arthritis ( ra ) , type 1 diabetes mellitus ( t1 dm ) , ankylosing spondylitis ( as ) , and preeclampsia ( pe ) . based on our results , 
 a number of dominant features were identified that were present in most of the investigated autoimmune diseases .