BACKGROUND
renal transplantation is used to improve survival and quality of life for patients with end-stage renal disease.
in the past, patients often eventually die from complications  <cit>  if toxins cannot be removed from the body by hemodialysis.
although renal transplantation is recognized as the gold strategy for treating renal failure, it has several limitations including donor’s immune rejection.
in order to identify a means of controlling immune rejection, further illustration on the mechanism of immune rejection in renal transplant recipients  has great significance.
it is generally accepted that a significant barrier to organ transplantation is the humoral and cellular rejection that can occur and mediated by antibodies, t cells, and innate immune cells.
cellular immune response play’s an equally important role with humoral immune response in allograft rejection  <cit> .
for instance, there is evidence that a disturbed t-cell homeostasis play’s a critical role in the development of acute graft rejection episodes.
the main t subsets which are pivotal for this t-cell balance consists of t-helper  <dig>  cells and regulatory t  cells .
in addition to well characterized th <dig> and th <dig> lymphocytes, additional subsets called th <dig> cells, which selectively produce il- <dig>  have joined the effector cd4+ t cell lineage.
imbalanced th <dig> and impaired treg cells have suggested to be involved in the pathogenesis of allograft rejection, such as heart and lung transplantations .
previous studies have suggested that th <dig> cells are important for clearance of a variety of pathogens and are associated with numerous autoimmune and inflammatory conditions  <cit> .
in addition, th <dig> cells have also been implicated in acute and chronic rejection in animal models of allograft transplant .
interestingly, the function of self-reacting effector th <dig> cells is controlled by tregs, yet another subpopulation of cd4+ t lymphocytes which express transcription factor foxp <dig>  <cit> .
tregs are important regulators of immune tolerance and can actively suppress pro-inflammatory t cell responses  <cit> .
quantitative and/or qualitative deficiencies of tregs have been associated with the development of organ transplantation rejection .
previous studies in animal models have shown that a deficiency in tregs favors kidney transplantation rejection  <cit> , though their mechanism in clinical studies remains unclear.
human tregs are not as well characterized as their murine counterparts; in part this is due to restrictions and limitations of clinical studies.
furthermore, the characterization of tregs in humans is more complex  <cit> .
human tregs are cd4+cd25+ and their development and function depends on the forkhead family transcription factor  expression .
recent study has shown that a lower frequency of circulating cd4+cd25+foxp3+ t cells was detected in rtr patients, and the percentages of cd4+cd25+foxp3+ t cells were negatively associated with egfr of rtr  <cit> .
however, little is known about the number of tregs and th <dig> cells, and their association with different types of rejection in rtr patients.
in addition, studies have shown that some inflammatory cytokines, such as th1-type cytokine  and th17-type cytokine , are also associated with the development of rejection .
for instance, ifn-γ can mediate separate functions at the same target organ during graft-versus-host disease , and il- <dig> can induce the expression of proinflammatory tumor necrosis factor -α, chemotactic protein - <dig> and macrophage inflammatory protein - <dig> to promote tissue inflammation .
furthermore, il- <dig> can also promote the differentiation and maturation of dendritic precursor cells, increased cell surface expression of cd <dig>  cd <dig> and major histocompatibility complex -ii antigen  <cit> .
however, the role of these inflammatory cytokines in different types of renal transplantation rejection has not been clarified.
in the current study, we characterized the number of circulating cd4+cd25+foxp3+ tregs and th <dig> cells, and the concentration of serum inflammatory cytokines in different rtr patients and hcs to determine their potential association with clinical measures in the patients.
methods
subjects
a total of  <dig> patients , 31– <dig> years of age , with an end-stage renal failure  waiting for renal transplantation were recruited from the inpatient service of the first hospital of jilin university, changchun, china.
ten gender, ethnicity and age-matched hcs were included in the study.
individual patients with esrf were diagnosed according to the criteria  <cit> .
individual patients were treated with conventional immunosuppressors  twice a day for three days.
all patients had compatible hla-gene matches and the number of those patients who had hla  mismatches were less than two.
individuals were excluded if she/he had a history of previous renal transplantation or surgical procedure.
written informed consent was obtained from individual participants.
the experimental protocol was established according to the guidelines of the  <dig> declaration of helsinki and approved by the human ethics committee of jilin university, china.
study groups
transplant patients were divided into four groups according to graft function  or serum creatinine level) and post-transplant rejection time  as well as the banff classification  <cit> .
the four groups included were:  transplant stable  : recipients with stable graft function under conventional immunosuppressors  and without clinical and laboratory features suggestive of rejection  in 12 weeks; there was no biopsies of these individuals because they have normal and stable graft function;  acute antibody mediated acute rejection  : recipients under conventional immunosuppressors with clinical symptoms  and progressive renal function deterioration  during 1 week; the rejection of these individuals was confirmed by biopsy according to banff criteria;  acute rejection  : recipients under conventional immunosuppressors with clinical symptoms  and progressive renal function deterioration  during  <dig> to 12 weeks; the rejection of these individuals was confirmed by biopsy according to banff criteria;  chronic rejection  : recipients under conventional immunosuppressors with clinical symptoms  and progressive renal function deterioration  after 12 weeks; the rejection of these individuals was confirmed by biopsy according to banff criteria.
these group of patients who received kidney transplant had no post-transplant malignancy and infective complications.
in case of rejection response, patients withdrew from the follow-up and receive additional anti-rejection treatment, such as methyl prednisolone, anti-thymocyte globulin or anti-cd <dig> monoclonal antibody as well as plasma exchange.
clinical measurement
peripheral venous blood samples were obtained from individual participants for laboratory test before and after transplant, when rejection-related clinical symptoms appeared.
the routine laboratory investigations include complete blood count, serum creatinine, bun and glomerular filtration rate .
the laboratory investigations were conducted by scattered turbidimetry using siemens special protein analyzer .
isolation and stimulation of pbmcs
peripheral venous blood samples were collected after overnight fasting.
peripheral blood mononuclear cells  were isolated by density-gradient centrifugation using ficoll-paque plus .
pbmcs  were stimulated in duplicate with 50 ng/ml of phorbol myristate acetate  and  <dig>  mg/ml of ionomycin  in 10 % human ab type sera in rpmi  <dig> medium at room temperature in a humidified incubator with 95 % air and 5 % carbon dioxide for 2 h and then cultured for another 4 h in the presence of  <dig>  mg/ml of brefeldin a .
the control pbmcs were cultured in medium alone.
flow cytometry analysis
the frequency of cd4+ifn-γ+il-17− th <dig>  cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17+ th1/ <dig> and cd4+cd25+foxp3+ t cells in individual samples were determined by flow cytometry following intracellular staining with anti-cytokine antibodies.
briefly, the stimulated pbmcs were harvested and stained with allophycocyanin -labeled anti-cd <dig>  fixed with the perm/fix solution, and permeabilized, followed by staining with fluorescein isothiocyanate -labeled anti–il- <dig> and pe-cy7-labeled anti-ifn-γ .
additional cells were stained in duplicate with percp-anti-cd4/fitc-anti-cd <dig> or isotype-matched controls  for 30 min, fixed, and permeabilized using the permeabilization solution , followed by intracellular staining with pe-anti-poxp- <dig> .
after being washed with pbs, these cells were analyzed on a facscalibur  and at least  <dig>  events were analyzed by flowjo software .
cytometric bead array for the level of serum cytokines
the concentrations of serum ifn-γ, tnf-α, il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig> were determined by cytometric bead array , according to the manufacturer’s protocol  with minor modification.
briefly, 50 μl serum samples were subjected to analysis in duplicate using the cytometric bead array kit on a facscalibur cytometry.
the concentration of serum cytokines were quantified using cellquestpro and cba software .
the detection limit for ifn-γ, tnf-α, il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig> were  <dig>  pg/ml,  <dig>  pg/ml,  <dig>  pg/ml,  <dig>  pg/ml,  <dig>  pg/ml,  <dig>  pg/ml, and  <dig>  pg/ml, respectively.
statistical analysis
data were expressed as median and range for each group unless specified.
the difference between groups was analyzed by the kruskal-wallis test or chi-square test using spss  <dig>  software for unpaired and paired comparisons, respectively.
the relationship between variables was evaluated using the spearman rank correlation test.
a two-side p value <  <dig>  was considered statistically significant.
RESULTS
patient sociodemographic and clinical characteristics
the sociodemographic and clinical characteristics of rtr’s are summarized in table  <dig>  the patients displayed higher concentration of serum creatinine and bun and lower level of egfr.
furthermore, significantly higher levels of white blood cell , and lower levels of red blood cell  and hemoglobin  were detected in esrf, compared to the hcs.table  <dig> the demographic and clinical characteristics of subjects
data shown are median  of each group of subjects
esrf end-stage renal failure, bun blood urea nitrogen , cr serum creatinine , egfr glomerular filtration rate , wbc white blood cell , rbc: normal range: men,  <dig>  ~  <dig>  × 1012/l; women,  <dig>  ~  <dig>  × 1012/l; hb: normal range: men, 120-160 g/l; women, 110–150 g/l
*p <  <dig>  vs. the hc
rtr’s were divided into four groups according to graft function  and post-transplant rejection time .
ts patients showed a significant reduction in bun and cr levels and a significant increase in egfr values and hb compared to the pre transplant status.
in addition, the concentration of serum cr and bun in amr, ar and cr groups were significantly higher than ts esrf patients.table  <dig> clinical characteristics of before and post-transplant patients
data are expressed as median  or real case numbers
* p <  <dig>  vs, before transplant rejection
cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/17 t cells in the patients
the circulating cd4+ t cells analysis  found that, the number of cd4+, cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells  in patients with esrf were significantly greater than the hcs.
in contrast, there was no significant difference in the number of cd4+cd25+foxp3+ t cells  between the esrf patients and hcs.
as a result, the ratio of cd4+cd25+foxp3+ t cells to cd4+ifn-γ−il-17+ th <dig> cells  was significantly lower in esrf patients compared to the hcs.
furthermore, there was no significant difference in the ratio of cd4+cd25+foxp3+ t cells to cd4+ifn-γ+il-17− th <dig> or cd4+ifn-γ+il-17+ th1/ <dig> cells between esrf patients and the hcs.fig.
<dig> facs analysis of circulating cd4+ t cells.
pbmcs from individual esrf patients and hc subjects were stained with apc-anti-cd <dig>  or percp-anti-cd <dig> and fitc-anti-cd <dig>  or isotype controls, fixed and permeabilized, followed by intracellular staining with fitc-anti–il- <dig> and pe-cy7-anti-ifn-γ and pe-anti-foxp <dig>  the frequency of cd4+, cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells were determined by flow cytometry analysis.
the cells were gated on living lymphocytes and then gated on cd4+ cells, and at least about  <dig>  events were analyzed for each sample.
the numbers of each type of cd4+ t cells were calculated, according to the total numbers of pbmcs, the frequency of total cd4+, and different types of cd4+ t cells.
a the representative charts of flow cytometry analysis.
b-g quantitative analysis.
data shown are representative facs charts or the mean numbers of each type of cells per ml of peripheral blood in individual subjects from two separate experiments.
the horizontal lines indicate the median values for each group
the number of cd4 + ifn-γ-il-17+ th <dig>  cd4 + ifn-γ + il-17- th <dig>  cd4 + ifn-γ + il-17+ th1/17 t and cd4 + cd25 + foxp3+ t cells
further comparison of different types of cd4+ t cells  found that, in post-transplant ts patients, the number of cd4+cd25+foxp3+ t cells and the ratio of tregs to th <dig> were significantly increased , whereas the number of cd4+, cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells were significantly decreased compared to the pre-transplant status.
.
conversely, in the amr, ar and cr patients, the number of cd4+cd25+foxp3+ t cells is decreased while the numbers of cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells are increased .
moreover, there was no significant change in the ratio of tregs to th <dig> cells in the pre- and post-transplant status.
interestingly, amr patients displayed higher number of cd4+, cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells and lower number of cd4+cd25+foxp3+ t cells compared to ar and cr patients.
these data suggested that altered cd <dig> + t cells number may be associated with renal transplantation rejection.fig.
<dig> stratification analysis of the numbers of different types of cd4+ t cells in different groups of renal transplant patients.
the patients were stratified into the transplant stable  , accelerated rejection  , sub-acute rejection   and chronic rejection   groups, the numbers of cd4+, cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells and the ratios of tregs to th <dig> cells in these four groups of patients were analyzed.
a the representative charts of flow cytometry analysis.
b-g quantitative analysis.
data shown are representative facs charts or the mean numbers of each type of cells per ml of peripheral blood in individual subjects from two separate experiments.
the horizontal lines indicate the median values for each group
serum th1- and th17-type cytokines in rtr patients
the concentration of serum il- <dig>  ifn-γ, tnf-α, il- <dig>  il- <dig>  il- <dig> and il- <dig> in esrf patients were significantly increased compared to the hc’s .
the serum cytokines level were determined and found significantly increased serum levels of il- <dig>  ifn-γ, tnf-α and il- <dig> in post-transplant ts’s compared to the pre-transplant patients.
ts patients showed an increased level of serum il- <dig> compared to the pre-transplant rtr’s .
however, there was no significant difference in the concentrations of il- <dig> and il- <dig> pre and post-transplant.
these data indicated that higher serum levels of th17- and th1-type cytokines may be associated with renal transplantation rejection.fig.
<dig> the levels of serum inflammatory cytokines in esrf and rtr patients.
a the levels of serum ifn-γ, tnf-α, il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig> in hcs and esrf patients.
b plasma levels of serum ifn-γ, tnf-α, il- <dig>  il- <dig>  il- <dig>  il- <dig> and il- <dig> in patients at before and post-transplant in these groups of patients were analyzed.
the horizontal lines indicate the median values of the different groups
the correlation of tregs with th <dig> cells in rtr patients
the potential relationship between the concentration of serum inflammatory cytokines and the number of different subsets of cd4+ t cells in rtr patients was analyzed .
it was found that, the concentration of serum il- <dig> and ifn-γ was positively correlated with the number of cd4+ifn-γ−il-17+ th <dig> and cd4+ifn-γ+il-17− th <dig> cells, respectively, in the four groups.
further analysis of the relationship found a negative association between the number of cd4+cd25+foxp3+ t cells and the number of cd4+ifn-γ−il-17+ th <dig> cells in the four groups.
these data suggested that different types of cd4+ t cells may have variable functions during renal transplantation rejection.fig.
<dig> correlation between the numbers of circulating cd4+ t cells and serum levels of inflammatory cytokines in rtr patients.
correlation between the numbers of cd4+cd25+foxp3+ t, cd4+ifn-γ−il-17+ th <dig> and cd4+ifn-γ+il-17− th <dig> cells and cd4+ifn-γ−il-17+ th <dig> cells, serum levels of il- <dig> and ifn-γ in ts , amr , ar  and cr  patients
the relationship between the number of cd4+ t cells or inflammatory cytokines and clinical parameters in ts patients
to understand the importance of different subsets of cd4+ t cells and inflammatory cytokines, we analyzed the potential association of the number of different types of cd4+ t cells and inflammatory cytokines with clinical parameters of the patients.
it was found that the number of cd4 + cd25 + foxp3+ and cd4 + ifn-γ-il-17+ th <dig> cells were positively correlated with the egfr value and serum creatinine level, respectively, in the ts group .
further analysis revealed that the serum level of tnf-α and il- <dig> were positively correlated with serum creatinine level.
in contrast, the serum level of il- <dig> was negatively correlated with serum creatinine in the ts group.
moreover, three groups of rtr also showed the same trend .fig.
<dig> correlation between the numbers of circulating cd4+ t cells or serum levels of inflammatory cytokines and disease activity in rtr patients.
correlation between the numbers of cd4+cd25+foxp3+ t, cd4+ifn-γ−il-17+ th <dig> cells and serum levels of tnf-α, il- <dig> and il- <dig> and the valves of egfr and the serum levels of cr in ts patients
discussion
although renal transplantation provides a readily accessible alternative strategy for patients with end-stage renal failure, immune rejection remains a major hurdle to its implementation.
in this study, we examined the pre and post-transplantation number of different types of circulating cd4+ t cells in esrf patients.
the study found that, the number of cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells in esrf patients were significantly increased compared to the hc, whereas there was no significant difference in the number of cd4+cd25+foxp3+ t cells between patients and hc’s.
apparently, there was an imbalance of tregs to th <dig> cells in esrf patients.
together these data suggested that dysregulated cd4+ t cells play an important role in esrf patients, which were consistent with previous studies  <cit> .
moreover, rtr showed increased number of cd4+ifn-γ−il-17+ th <dig>  cd4+ifn-γ+il-17− th <dig> and cd4+ifn-γ+il-17+ th1/ <dig> cells and higher levels of serum il- <dig>  ifn-γ, tnf-α and il- <dig>  but decreased number of cd4+cd25+foxp3+ t cells and lower levels of serum il- <dig> compared to pre-transplant and ts’s patients.
conceivably, an imbalance in cd4+ t cells and inflammatory cytokines were new target for immunotherapy for the intervention of renal transplantation.
increasing evidences have shown that multiple subsets of effector cd4+ t cells play an important role in allograft rejection  <cit> .
in addition to direct killing activity by cytotoxic t lymphocytes, organ allotransplantation rejection can occur through t-cell-mediated mechanisms, including cytokine production, recruitment and activation of other cytotoxic cells as well as b cells that produce xenoreactive antibodies  <cit> .
this study demonstrated that after renal transplant, patients showed increased number of cd4+ifn-γ+il-17− th <dig> cells and th1-type cytokines  compared to the pre-transplant and ts patients, which was consistent with previous reports .
similarly, we detected significantly higher levels of cytokines in post-transplant patients compared to pre-transplant.
moreover, the number of cd4+ifn-γ+il-17− th <dig> cells was positively correlated with the concentration of serum ifn-γ in all the four post-transplant groups.
however, there was no significant difference in the th2-type cytokines  before and after transplant.
these data suggested that th <dig>  not th <dig>  likely play an important role in rejection, which were consistent with yuxin and coworkers  <cit> .
the significantly changed th <dig> cells may stem from the inflammatory environment, which preferably activate naive helper t cells towards th <dig> direction.
in addition to th <dig> cells, th <dig> cells expressing retinoic acid-related orphan receptor γt  that play a crucial role in the development of transplant rejection by producing pro-inflammatory cytokine il- <dig> .
some studies have demonstrated that th <dig> and il- <dig> contribute to rejection during heart, lung, liver and other organ allotransplantation  <cit> .
morever, similar studies have also suggested that il-17-secreting cell infiltration is a prognostic marker for determining allograft outcome in renal allograft biopsies with acute t-cell-mediated rejection   <cit> .
however, the possible mechanisms of th <dig> and il- <dig> in different types of renal transplantation rejection remains unknown.
notably, we detected significantly increased number of cd4+ifn-γ−il-17+ th <dig> cells and th17-type cytokines  in rtr compared to the pre-transplant status and ts patients.
moreover, the number of th <dig> cells was positively correlated with serum il- <dig> concentration in the post-transplant groups.
these results suggested that th <dig> cells, as well as th <dig> cells, play a key role in renal transplantation rejection.
more interestingly, the study found greater number of cd4+ifn-γ+il-17+ th1/ <dig> cells in rtr group compared to pre-transplant and post-transplant ts patients.
previous studies have shown that cd4+ifn-γ−il-17+ th <dig> cells are a rare population and the development of these cells depends on the cytokine environment .
however, there was no significant correlation between the number of th1/ <dig> cells and the clinical parameters.
it is possible that th1/ <dig> cells may not be potent effectors for transplant rejection.
given that, following activation, t cells can differentiate into different functional t cells, these th1/ <dig> cells may be early differentiated and uncommitted cells.
we are interested to further investigating the role of th1/ <dig> cells in the renal transplantation rejection.
similar studies have reported that cd4+cd25+foxp3+ t cells play an important role in immune tolerance mechanism during renal transplantation rejection .
a study by wen et al.
<cit>  found that following transplantation, rtr had significantly lower levels of cd4+cd25+foxp3+ t cells compared to esrf and these cells had a positive linear relationship with glomerular filtration rate.
this finding was consistent with our data that showed rtr had lower number of cd4+cd25+foxp3+ t cells compared to pre-transplant status.
this observation may partly reflect the impact of immunosuppression on this cell population.
studies of tregs in rtr have shown variably changing circulating levels during post-transplantation, suggesting the possibility of regulatory cells production expressing foxp <dig> in immunosuppressed rtr’s.
further studies on cd4+ t-cell subsets in rtr have indicated that the number of cd4+cd25+foxp3+ t cells were negatively correlated with the number of cd4+ifn-γ−il-17+ th <dig> cells, which was supported by molecular studies showing the presence of reciprocal interaction between these subpopulation.
though, both th <dig> and tregs require tgf-β <dig> during the early stage of differentiation, in the presence of pro-inflammatory cytokines tgf-β <dig> and il- <dig>  foxp <dig> is down-regulated and t-cell with transitional phenotype express a set of proteins essential for th <dig> development  <cit> .
additionally, il- <dig>  which is required for the regulation of foxp <dig> expressing tregs, has been found to inhibit the development of th <dig> cells  <cit> .
hence, it is possible that the counteractive effects of il- <dig> and il- <dig> on the differentiation of th <dig> and treg in the periphery may hamper immunoregulatory responses and facilitate the persistence of rejection.
morever, previous studies have suggested that the foxp3/il- <dig> ratio may be a useful indicator for representing the severity of tissue injury, allograft dys-function and for predicting the clinical outcome of atcmr  <cit> .
however, the precise roles of treg and th <dig> cells in renal transplantation rejection need to be further explored.
morever, our study showed that there was no correlation between the number of cd4+cd25+foxp3+ t cells and the number of cd4+ifn-γ+il-17− th <dig> cells, which was inconsistent with previous research that showed suppression of th <dig> cell-mediated responses by tregs through inhibition of monocyte-derived il- <dig>  <cit> .
this possibility could be attributed to the difference in the internal circumstances of the patients.
previous studies have reported that multiple inflammatory cytokines played a key role in transplantation rejection response , such as th1-type  and th17-type  cytokines.
il- <dig> is the critical mediator of acute rejection whereas ifn-γ can mediate separate functions at the same target organ during graft-versus-host disease  .
tnf-α is a lymphocyte and macrophage derived cytokine that is pleiotropic in its actions.
its proinflammatory function suggest that it may play an important role in initiating and orchestrating the rejection response.
studies demonstrating a correlation in the expression of tnf-alpha with the severity of the rejection episode have placed tnf-alpha as a prime candidate marker of transplantation rejection  <cit> .
il <dig> is a potent synthesis inhibitory factor and anti-inflammatory, and is an important immunoregulatory component in the cytokine network of rtr’s  <cit> .
il- <dig> can induce the expression of proinflammatory tnf-α, and mcp- <dig> and mip- <dig> to promote tissue inflammation  <cit> .
in the present study, we found that rtr patients displayed higher levels of serum il- <dig>  ifn-γ, tnf-α and il- <dig>  but not il- <dig> and il- <dig>  whereas ts patients displayed higher levels of serum il- <dig> compared to pre-transplant status.
more importantly, the serum levels of tnf-α and il- <dig> were positively correlated with the concentrations of serum cr, whereas il- <dig> level was negatively correlated with the concentration of serum cr in all the four post-transplant groups.
these data indicated that an imbalance of serum th1- and th17-type cytokines may be a positive regulator during renal transplantation rejection process.
conceivably, the change in inflammatory cytokines may be a new target for designing immunotherapy for the intervention of renal transplantation rejection response.
CONCLUSIONS
in summary, our data showed a significantly increased number of circulating th <dig> and th <dig> cells as well as higher levels of serum inflammatory cytokines in esrf patients.
rtr displayed lower number of tregs, treg to th <dig> cells ratio and serum il- <dig>  and higher numbers of th <dig> and th <dig> and related cytokines compared to the pre-transplant and post-transplant ts patients.
these novel finding suggested that effector cd4+ t cells may be controlled by tregs during renal transplantation rejection and the imbalance of treg/th <dig> axis may be associated with the process of renal transplantation rejection.
the limitations of this study include small sample size and lack of functional studies of different types of cd4+ t cells.
thus, further studies in larger population are warranted.
liang ma and huimao zhang contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
yj conceived the study.
lm performed the experiments and wrote the manuscript.
hz performed allmathematical and statistical analysis.
kh, gl and yf interpreted the analysis results and planned the validation experiments.
pz supervised the experiments.
all authors read and approved the final manuscript.

 renal transplantation is used to improve survival and quality of life for patients with end-stage renal disease. in the past, patients often eventually die from complications if toxins cannot be removed from the body by hemodialysis. although renal transplantation is recognized as the gold strategy for treating renal failure, it has several limitations including donor’s immune rejection. in order to identify a means of controlling immune rejection, further illustration on the mechanism of immune rejection in renal transplant recipients has great significance. it is generally accepted that a significant barrier to organ transplantation is the humoral and cellular rejection that can occur and mediated by antibodies, t cells, and innate immune cells. cellular immune response play’s an equally important role with humoral immune response in allograft rejection. for instance, there is evidence that a disturbed t-cell homeostasis play’s a critical role in the development of acute graft rejection episodes. the main t subsets which are pivotal for this t-cell balance consists of t-helper cells and regulatory t cells. in addition to well characterized th and th lymphocytes, additional subsets called th cells, which selectively produce il- have joined the effector cd4+ t cell lineage. imbalanced th and impaired treg cells have suggested to be involved in the pathogenesis of allograft rejection, such as heart and lung transplantations. previous studies have suggested that th cells are important for clearance of a variety of pathogens and are associated with numerous autoimmune and inflammatory conditions. in addition, th cells have also been implicated in acute and chronic rejection in animal models of allograft transplant. interestingly, the function of self-reacting effector th cells is controlled by tregs, yet another subpopulation of cd4+ t lymphocytes which express transcription factor foxp. tregs are important regulators of immune tolerance and can actively suppress pro-inflammatory t cell responses. quantitative and/or qualitative deficiencies of tregs have been associated with the development of organ transplantation rejection. previous studies in animal models have shown that a deficiency in tregs favors kidney transplantation rejection, though their mechanism in clinical studies remains unclear. human tregs are not as well characterized as their murine counterparts; in part this is due to restrictions and limitations of clinical studies. furthermore, the characterization of tregs in humans is more complex. human tregs are cd4+cd25+ and their development and function depends on the forkhead family transcription factor expression. recent study has shown that a lower frequency of circulating cd4+cd25+foxp3+ t cells was detected in rtr patients, and the percentages of cd4+cd25+foxp3+ t cells were negatively associated with egfr of rtr. however, little is known about the number of tregs and th cells, and their association with different types of rejection in rtr patients. in addition, studies have shown that some inflammatory cytokines, such as th1-type cytokine and th17-type cytokine, are also associated with the development of rejection. for instance, ifn-γ can mediate separate functions at the same target organ during graft-versus-host disease, and il- can induce the expression of proinflammatory tumor necrosis factor -α, chemotactic protein - and macrophage inflammatory protein - to promote tissue inflammation. furthermore, il- can also promote the differentiation and maturation of dendritic precursor cells, increased cell surface expression of cd cd and major histocompatibility complex -ii antigen. however, the role of these inflammatory cytokines in different types of renal transplantation rejection has not been clarified. in the current study, we characterized the number of circulating cd4+cd25+foxp3+ tregs and th cells, and the concentration of serum inflammatory cytokines in different rtr patients and hcs to determine their potential association with clinical measures in the patients. methods subjects a total of patients, 31– years of age, with an end-stage renal failure waiting for renal transplantation were recruited from the inpatient service of the first hospital of jilin university, changchun, china. ten gender, ethnicity and age-matched hcs were included in the study. individual patients with esrf were diagnosed according to the criteria. individual patients were treated with conventional immunosuppressors twice a day for three days. all patients had compatible hla-gene matches and the number of those patients who had hla mismatches were less than two. individuals were excluded if she/he had a history of previous renal transplantation or surgical procedure. written informed consent was obtained from individual participants. the experimental protocol was established according to the guidelines of the declaration of helsinki and approved by the human ethics committee of jilin university, china. study groups transplant patients were divided into four groups according to graft function or serum creatinine level) and post-transplant rejection time as well as the banff classification. the four groups included were: transplant stable: recipients with stable graft function under conventional immunosuppressors and without clinical and laboratory features suggestive of rejection in 12 weeks; there was no biopsies of these individuals because they have normal and stable graft function; acute antibody mediated acute rejection: recipients under conventional immunosuppressors with clinical symptoms and progressive renal function deterioration during 1 week; the rejection of these individuals was confirmed by biopsy according to banff criteria; acute rejection: recipients under conventional immunosuppressors with clinical symptoms and progressive renal function deterioration during to 12 weeks; the rejection of these individuals was confirmed by biopsy according to banff criteria; chronic rejection: recipients under conventional immunosuppressors with clinical symptoms and progressive renal function deterioration after 12 weeks; the rejection of these individuals was confirmed by biopsy according to banff criteria. these group of patients who received kidney transplant had no post-transplant malignancy and infective complications. in case of rejection response, patients withdrew from the follow-up and receive additional anti-rejection treatment, such as methyl prednisolone, anti-thymocyte globulin or anti-cd monoclonal antibody as well as plasma exchange. clinical measurement peripheral venous blood samples were obtained from individual participants for laboratory test before and after transplant, when rejection-related clinical symptoms appeared. the routine laboratory investigations include complete blood count, serum creatinine, bun and glomerular filtration rate. the laboratory investigations were conducted by scattered turbidimetry using siemens special protein analyzer. isolation and stimulation of pbmcs peripheral venous blood samples were collected after overnight fasting. peripheral blood mononuclear cells were isolated by density-gradient centrifugation using ficoll-paque plus. pbmcs were stimulated in duplicate with 50 ng/ml of phorbol myristate acetate and  mg/ml of ionomycin in 10 % human ab type sera in rpmi medium at room temperature in a humidified incubator with 95 % air and 5 % carbon dioxide for 2 h and then cultured for another 4 h in the presence of  mg/ml of brefeldin a. the control pbmcs were cultured in medium alone. flow cytometry analysis the frequency of cd4+ifn-γ+il-17− th cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17+ th1/ and cd4+cd25+foxp3+ t cells in individual samples were determined by flow cytometry following intracellular staining with anti-cytokine antibodies. briefly, the stimulated pbmcs were harvested and stained with allophycocyanin -labeled anti-cd fixed with the perm/fix solution, and permeabilized, followed by staining with fluorescein isothiocyanate -labeled anti–il- and pe-cy7-labeled anti-ifn-γ. additional cells were stained in duplicate with percp-anti-cd4/fitc-anti-cd or isotype-matched controls for 30 min, fixed, and permeabilized using the permeabilization solution, followed by intracellular staining with pe-anti-poxp-. after being washed with pbs, these cells were analyzed on a facscalibur and at least events were analyzed by flowjo software. cytometric bead array for the level of serum cytokines the concentrations of serum ifn-γ, tnf-α, il- il- il- il- and il- were determined by cytometric bead array, according to the manufacturer’s protocol with minor modification. briefly, 50 μl serum samples were subjected to analysis in duplicate using the cytometric bead array kit on a facscalibur cytometry. the concentration of serum cytokines were quantified using cellquestpro and cba software. the detection limit for ifn-γ, tnf-α, il- il- il- il- and il- were  pg/ml,  pg/ml,  pg/ml,  pg/ml,  pg/ml,  pg/ml, and  pg/ml, respectively. statistical analysis data were expressed as median and range for each group unless specified. the difference between groups was analyzed by the kruskal-wallis test or chi-square test using spss software for unpaired and paired comparisons, respectively. the relationship between variables was evaluated using the spearman rank correlation test. a two-side p value <  was considered statistically significant.  patient sociodemographic and clinical characteristics the sociodemographic and clinical characteristics of rtr’s are summarized in table  the patients displayed higher concentration of serum creatinine and bun and lower level of egfr. furthermore, significantly higher levels of white blood cell, and lower levels of red blood cell and hemoglobin were detected in esrf, compared to the hcs.table the demographic and clinical characteristics of subjects data shown are median of each group of subjects esrf end-stage renal failure, bun blood urea nitrogen, cr serum creatinine, egfr glomerular filtration rate, wbc white blood cell, rbc: normal range: men,  ~   × 1012/l; women,  ~   × 1012/l; hb: normal range: men, 120-160 g/l; women, 110–150 g/l *p <  vs. the hc rtr’s were divided into four groups according to graft function and post-transplant rejection time. ts patients showed a significant reduction in bun and cr levels and a significant increase in egfr values and hb compared to the pre transplant status. in addition, the concentration of serum cr and bun in amr, ar and cr groups were significantly higher than ts esrf patients.table clinical characteristics of before and post-transplant patients data are expressed as median or real case numbers * p <  vs, before transplant rejection cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/17 t cells in the patients the circulating cd4+ t cells analysis found that, the number of cd4+, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells in patients with esrf were significantly greater than the hcs. in contrast, there was no significant difference in the number of cd4+cd25+foxp3+ t cells between the esrf patients and hcs. as a result, the ratio of cd4+cd25+foxp3+ t cells to cd4+ifn-γ−il-17+ th cells was significantly lower in esrf patients compared to the hcs. furthermore, there was no significant difference in the ratio of cd4+cd25+foxp3+ t cells to cd4+ifn-γ+il-17− th or cd4+ifn-γ+il-17+ th1/ cells between esrf patients and the hcs.fig. facs analysis of circulating cd4+ t cells. pbmcs from individual esrf patients and hc subjects were stained with apc-anti-cd or percp-anti-cd and fitc-anti-cd or isotype controls, fixed and permeabilized, followed by intracellular staining with fitc-anti–il- and pe-cy7-anti-ifn-γ and pe-anti-foxp the frequency of cd4+, cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells were determined by flow cytometry analysis. the cells were gated on living lymphocytes and then gated on cd4+ cells, and at least about events were analyzed for each sample. the numbers of each type of cd4+ t cells were calculated, according to the total numbers of pbmcs, the frequency of total cd4+, and different types of cd4+ t cells. a the representative charts of flow cytometry analysis. b-g quantitative analysis. data shown are representative facs charts or the mean numbers of each type of cells per ml of peripheral blood in individual subjects from two separate experiments. the horizontal lines indicate the median values for each group the number of cd4 + ifn-γ-il-17+ th cd4 + ifn-γ + il-17- th cd4 + ifn-γ + il-17+ th1/17 t and cd4 + cd25 + foxp3+ t cells further comparison of different types of cd4+ t cells found that, in post-transplant ts patients, the number of cd4+cd25+foxp3+ t cells and the ratio of tregs to th were significantly increased, whereas the number of cd4+, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells were significantly decreased compared to the pre-transplant status.. conversely, in the amr, ar and cr patients, the number of cd4+cd25+foxp3+ t cells is decreased while the numbers of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells are increased. moreover, there was no significant change in the ratio of tregs to th cells in the pre- and post-transplant status. interestingly, amr patients displayed higher number of cd4+, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells and lower number of cd4+cd25+foxp3+ t cells compared to ar and cr patients. these data suggested that altered cd + t cells number may be associated with renal transplantation rejection.fig. stratification analysis of the numbers of different types of cd4+ t cells in different groups of renal transplant patients. the patients were stratified into the transplant stable, accelerated rejection, sub-acute rejection and chronic rejection groups, the numbers of cd4+, cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells and the ratios of tregs to th cells in these four groups of patients were analyzed. a the representative charts of flow cytometry analysis. b-g quantitative analysis. data shown are representative facs charts or the mean numbers of each type of cells per ml of peripheral blood in individual subjects from two separate experiments. the horizontal lines indicate the median values for each group serum th1- and th17-type cytokines in rtr patients the concentration of serum il- ifn-γ, tnf-α, il- il- il- and il- in esrf patients were significantly increased compared to the hc’s. the serum cytokines level were determined and found significantly increased serum levels of il- ifn-γ, tnf-α and il- in post-transplant ts’s compared to the pre-transplant patients. ts patients showed an increased level of serum il- compared to the pre-transplant rtr’s. however, there was no significant difference in the concentrations of il- and il- pre and post-transplant. these data indicated that higher serum levels of th17- and th1-type cytokines may be associated with renal transplantation rejection.fig. the levels of serum inflammatory cytokines in esrf and rtr patients. a the levels of serum ifn-γ, tnf-α, il- il- il- il- and il- in hcs and esrf patients. b plasma levels of serum ifn-γ, tnf-α, il- il- il- il- and il- in patients at before and post-transplant in these groups of patients were analyzed. the horizontal lines indicate the median values of the different groups the correlation of tregs with th cells in rtr patients the potential relationship between the concentration of serum inflammatory cytokines and the number of different subsets of cd4+ t cells in rtr patients was analyzed. it was found that, the concentration of serum il- and ifn-γ was positively correlated with the number of cd4+ifn-γ−il-17+ th and cd4+ifn-γ+il-17− th cells, respectively, in the four groups. further analysis of the relationship found a negative association between the number of cd4+cd25+foxp3+ t cells and the number of cd4+ifn-γ−il-17+ th cells in the four groups. these data suggested that different types of cd4+ t cells may have variable functions during renal transplantation rejection.fig. correlation between the numbers of circulating cd4+ t cells and serum levels of inflammatory cytokines in rtr patients. correlation between the numbers of cd4+cd25+foxp3+ t, cd4+ifn-γ−il-17+ th and cd4+ifn-γ+il-17− th cells and cd4+ifn-γ−il-17+ th cells, serum levels of il- and ifn-γ in ts, amr, ar and cr patients the relationship between the number of cd4+ t cells or inflammatory cytokines and clinical parameters in ts patients to understand the importance of different subsets of cd4+ t cells and inflammatory cytokines, we analyzed the potential association of the number of different types of cd4+ t cells and inflammatory cytokines with clinical parameters of the patients. it was found that the number of cd4 + cd25 + foxp3+ and cd4 + ifn-γ-il-17+ th cells were positively correlated with the egfr value and serum creatinine level, respectively, in the ts group. further analysis revealed that the serum level of tnf-α and il- were positively correlated with serum creatinine level. in contrast, the serum level of il- was negatively correlated with serum creatinine in the ts group. moreover, three groups of rtr also showed the same trend.fig. correlation between the numbers of circulating cd4+ t cells or serum levels of inflammatory cytokines and disease activity in rtr patients. correlation between the numbers of cd4+cd25+foxp3+ t, cd4+ifn-γ−il-17+ th cells and serum levels of tnf-α, il- and il- and the valves of egfr and the serum levels of cr in ts patients discussion although renal transplantation provides a readily accessible alternative strategy for patients with end-stage renal failure, immune rejection remains a major hurdle to its implementation. in this study, we examined the pre and post-transplantation number of different types of circulating cd4+ t cells in esrf patients. the study found that, the number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells in esrf patients were significantly increased compared to the hc, whereas there was no significant difference in the number of cd4+cd25+foxp3+ t cells between patients and hc’s. apparently, there was an imbalance of tregs to th cells in esrf patients. together these data suggested that dysregulated cd4+ t cells play an important role in esrf patients, which were consistent with previous studies. moreover, rtr showed increased number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells and higher levels of serum il- ifn-γ, tnf-α and il- but decreased number of cd4+cd25+foxp3+ t cells and lower levels of serum il- compared to pre-transplant and ts’s patients. conceivably, an imbalance in cd4+ t cells and inflammatory cytokines were new target for immunotherapy for the intervention of renal transplantation. increasing evidences have shown that multiple subsets of effector cd4+ t cells play an important role in allograft rejection. in addition to direct killing activity by cytotoxic t lymphocytes, organ allotransplantation rejection can occur through t-cell-mediated mechanisms, including cytokine production, recruitment and activation of other cytotoxic cells as well as b cells that produce xenoreactive antibodies. this study demonstrated that after renal transplant, patients showed increased number of cd4+ifn-γ+il-17− th cells and th1-type cytokines compared to the pre-transplant and ts patients, which was consistent with previous reports. similarly, we detected significantly higher levels of cytokines in post-transplant patients compared to pre-transplant. moreover, the number of cd4+ifn-γ+il-17− th cells was positively correlated with the concentration of serum ifn-γ in all the four post-transplant groups. however, there was no significant difference in the th2-type cytokines before and after transplant. these data suggested that th not th likely play an important role in rejection, which were consistent with yuxin and coworkers. the significantly changed th cells may stem from the inflammatory environment, which preferably activate naive helper t cells towards th direction. in addition to th cells, th cells expressing retinoic acid-related orphan receptor γt that play a crucial role in the development of transplant rejection by producing pro-inflammatory cytokine il-. some studies have demonstrated that th and il- contribute to rejection during heart, lung, liver and other organ allotransplantation. morever, similar studies have also suggested that il-17-secreting cell infiltration is a prognostic marker for determining allograft outcome in renal allograft biopsies with acute t-cell-mediated rejection. however, the possible mechanisms of th and il- in different types of renal transplantation rejection remains unknown. notably, we detected significantly increased number of cd4+ifn-γ−il-17+ th cells and th17-type cytokines in rtr compared to the pre-transplant status and ts patients. moreover, the number of th cells was positively correlated with serum il- concentration in the post-transplant groups. these results suggested that th cells, as well as th cells, play a key role in renal transplantation rejection. more interestingly, the study found greater number of cd4+ifn-γ+il-17+ th1/ cells in rtr group compared to pre-transplant and post-transplant ts patients. previous studies have shown that cd4+ifn-γ−il-17+ th cells are a rare population and the development of these cells depends on the cytokine environment. however, there was no significant correlation between the number of th1/ cells and the clinical parameters. it is possible that th1/ cells may not be potent effectors for transplant rejection. given that, following activation, t cells can differentiate into different functional t cells, these th1/ cells may be early differentiated and uncommitted cells. we are interested to further investigating the role of th1/ cells in the renal transplantation rejection. similar studies have reported that cd4+cd25+foxp3+ t cells play an important role in immune tolerance mechanism during renal transplantation rejection. a study by wen et al. found that following transplantation, rtr had significantly lower levels of cd4+cd25+foxp3+ t cells compared to esrf and these cells had a positive linear relationship with glomerular filtration rate. this finding was consistent with our data that showed rtr had lower number of cd4+cd25+foxp3+ t cells compared to pre-transplant status. this observation may partly reflect the impact of immunosuppression on this cell population. studies of tregs in rtr have shown variably changing circulating levels during post-transplantation, suggesting the possibility of regulatory cells production expressing foxp in immunosuppressed rtr’s. further studies on cd4+ t-cell subsets in rtr have indicated that the number of cd4+cd25+foxp3+ t cells were negatively correlated with the number of cd4+ifn-γ−il-17+ th cells, which was supported by molecular studies showing the presence of reciprocal interaction between these subpopulation. though, both th and tregs require tgf-β during the early stage of differentiation, in the presence of pro-inflammatory cytokines tgf-β and il- foxp is down-regulated and t-cell with transitional phenotype express a set of proteins essential for th development. additionally, il- which is required for the regulation of foxp expressing tregs, has been found to inhibit the development of th cells. hence, it is possible that the counteractive effects of il- and il- on the differentiation of th and treg in the periphery may hamper immunoregulatory responses and facilitate the persistence of rejection. morever, previous studies have suggested that the foxp3/il- ratio may be a useful indicator for representing the severity of tissue injury, allograft dys-function and for predicting the clinical outcome of atcmr. however, the precise roles of treg and th cells in renal transplantation rejection need to be further explored. morever, our study showed that there was no correlation between the number of cd4+cd25+foxp3+ t cells and the number of cd4+ifn-γ+il-17− th cells, which was inconsistent with previous research that showed suppression of th cell-mediated responses by tregs through inhibition of monocyte-derived il-. this possibility could be attributed to the difference in the internal circumstances of the patients. previous studies have reported that multiple inflammatory cytokines played a key role in transplantation rejection response, such as th1-type and th17-type cytokines. il- is the critical mediator of acute rejection whereas ifn-γ can mediate separate functions at the same target organ during graft-versus-host disease. tnf-α is a lymphocyte and macrophage derived cytokine that is pleiotropic in its actions. its proinflammatory function suggest that it may play an important role in initiating and orchestrating the rejection response. studies demonstrating a correlation in the expression of tnf-alpha with the severity of the rejection episode have placed tnf-alpha as a prime candidate marker of transplantation rejection. il is a potent synthesis inhibitory factor and anti-inflammatory, and is an important immunoregulatory component in the cytokine network of rtr’s. il- can induce the expression of proinflammatory tnf-α, and mcp- and mip- to promote tissue inflammation. in the present study, we found that rtr patients displayed higher levels of serum il- ifn-γ, tnf-α and il- but not il- and il- whereas ts patients displayed higher levels of serum il- compared to pre-transplant status. more importantly, the serum levels of tnf-α and il- were positively correlated with the concentrations of serum cr, whereas il- level was negatively correlated with the concentration of serum cr in all the four post-transplant groups. these data indicated that an imbalance of serum th1- and th17-type cytokines may be a positive regulator during renal transplantation rejection process. conceivably, the change in inflammatory cytokines may be a new target for designing immunotherapy for the intervention of renal transplantation rejection response.  in summary, our data showed a significantly increased number of circulating th and th cells as well as higher levels of serum inflammatory cytokines in esrf patients. rtr displayed lower number of tregs, treg to th cells ratio and serum il- and higher numbers of th and th and related cytokines compared to the pre-transplant and post-transplant ts patients. these novel finding suggested that effector cd4+ t cells may be controlled by tregs during renal transplantation rejection and the imbalance of treg/th axis may be associated with the process of renal transplantation rejection. the limitations of this study include small sample size and lack of functional studies of different types of cd4+ t cells. thus, further studies in larger population are warranted. liang ma and huimao zhang contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions yj conceived the study. lm performed the experiments and wrote the manuscript. hz performed allmathematical and statistical analysis. kh, gl and yf interpreted the analysis results and planned the validation experiments. pz supervised the experiments. all authors read and approved the final manuscript. 
stratification analysis indicated that amr, ar and cr groups displayed greater number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells as well as high level of serum il- ifn-γ, tnf-α and il- but, the amr, ar and cr groups have shown lower level of cd4+cd25+foxp3+ t cells and serum il- compared to transplant stable patients. a significant barrier to organ transplantation is the cellular rejection that occurs and mediated by antibodies, t cells, and innate immune cells. the imbalance between different types of cd4+ t cells and dysregulated inflammatory cytokines may contribute towards renal transplantation rejection. the number of cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells, and the serum level of ifn-γ, tnf-α, il- il- il- il- and il- were measured in pre- and post-transplant patients and healthy controls using flow cytometry and cytometric bead array. renal transplantation was performed for a total of patients with end-stage renal failure. the number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells were significantly increased in patients with end-stage renal failure compared to the hc. this study was aimed to determine the number of cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells in renal transplant recipients. the association between the number of different subsets of cd4+ t-cells and clinical parameters were analyzed among the pre- and post-transplant patients, and the healthy controls. moreover, the number of tregs were negatively correlated with the number of th cells in rtr patients.
 a significant barrier to organ transplantation is the cellular rejection that occurs and mediated by antibodies, t cells, and innate immune cells. this study was aimed to determine the number of cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells in renal transplant recipients. methods renal transplantation was performed for a total of patients with end-stage renal failure. the number of cd4+cd25+foxp3+ treg, cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells, and the serum level of ifn-γ, tnf-α, il- il- il- il- and il- were measured in pre- and post-transplant patients and healthy controls using flow cytometry and cytometric bead array. the association between the number of different subsets of cd4+ t-cells and clinical parameters were analyzed among the pre- and post-transplant patients, and the healthy controls.  the number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells were significantly increased in patients with end-stage renal failure compared to the hc. stratification analysis indicated that amr, ar and cr groups displayed greater number of cd4+ifn-γ−il-17+ th cd4+ifn-γ+il-17− th and cd4+ifn-γ+il-17+ th1/ cells as well as high level of serum il- ifn-γ, tnf-α and il- but, the amr, ar and cr groups have shown lower level of cd4+cd25+foxp3+ t cells and serum il- compared to transplant stable patients. moreover, the number of tregs were negatively correlated with the number of th cells in rtr patients. the number of tregs and th cells were positively correlated with the egfr and serum creatinine values, respectively.  the imbalance between different types of cd4+ t cells and dysregulated inflammatory cytokines may contribute towards renal transplantation rejection. issue-copyright-statement© the author 2015 