ra is an autoimmune disorder characterized by chronic inflammation of the synovial joints leading to destruction of cartilage , bone , and ligament .
the conventional treatment of ra with disease modifying antirheumatic drugs ( dmards ) aims to alleviate disease symptoms and delay or prevent future joint destruction .
advanced therapies are targeting inflammatory cytokines and autoreactive t cells , since the imbalance of inflammatory / anti - inflammatory cytokines is an important factor in ra pathogenesis .
dendritic cells ( dc ) are professional antigen - presenting cells ( apc ) and have an important role in both innate and adaptive immunity .
they are crucial for mediating immune system activation via antigen uptake and presentation to t cells .
dendritic cells also mediate immune tolerance to self - antigens and prevent excessive immune response [ 35 ] .
the basic knowledge surrounding tolerance induction has been greatly strengthened by the studies of several groups that generated human tolerogenic dcs ( toldcs ) in vitro [ 68 ] .
toldcs contribute to tolerance restoration through the induction of activation - induced cell death , anergy , and/or regulatory t cells in autoimmune diseases [ 9 , 10 ] .
a clinical trial that used toldcs to treat type i diabetes showed no severe adverse effects and opened the possibility of toldc use to treat autoimmune disease . although it remains unclear whether dcs have a role in the initiation of ra pathogenesis , evidence points toward a significant role for dcs in the maintenance and progression of ra . nevertheless , emerging therapies for ra are exploiting the tolerogenic capacity of dcs .
there are several clinical trials in progress that have exhibited that dc therapy is safe for the treatment of ra [ 12 , 13 ] .
we previously reported that semimature dcs ( smdcs ) have shown tolerogenic potential and a preventive effect when inoculated at a low dose in mice with collagen - induced arthritis ( cia ) .
however , the effect of smdcs or other toldcs has never been demonstrated in an advanced ra model . in the present study
, we evaluate the therapeutic effect of smdcs in combination with methotrexate ( mtx ) in advanced cia mice .
mtx is a first line dmard in most ra patients since mtx has a great efficacy / toxicity ratio .
it is a typical folic acid antagonist and also has an antirheumatic effect by antiproliferative , anti - inflammatory , and immunosuppressive mechanisms .
it has been reported that mtx treatment inhibits tnf- production and correlates with prevention of disease progression in a t cell - dependent mouse cia model [ 18 , 19 ] .
a possible mechanism is partial induction of regulatory t cells ( treg ) , induction of th1-to - th2 shift , and downregulation of th1 cytokines [ 16 , 20 ] .
however , the effect of mtx in an advanced cia model has never been reported . in the present study
, we show the therapeutic effect of smdcs in combination with mtx in an advanced cia model . here
, we found that combination therapy with smdcs and low - dose mtx was effective in controlling disease progression in advanced cia mice .
the benefit was likely associated with reduction of antigen - specific th1 and th17 populations in the spleen and an increase in the ln treg population after treatment .
this data will help us in our pursuit of using smdc in the treatment of ra , either with or without mtx .
six- to eight - week - old pathogen - free female dba/1j mice were purchased from charles river japan ( atsugi , kanagawa , japan ) and maintained in the animal facility of the jw creagene research institute ( gyeonggi , republic of korea ) .
all experiments were conducted in accordance with local animal ethics guidelines and approved by the institutional animal care and use committee ( iacuc ) .
cells were cultured in rpmi 1640 ( gibco laboratories , grand island , ny , usa ) supplemented with 10% fetal bovine serum , 50  nm 2-mercaptoethanol ( life technologies , gaithersburg , md , usa ) , 100  g / ml streptomycin , and 100  u / ml penicillin .
recombinant murine granulocyte - macrophage colony - stimulating factor ( gm - csf ) and recombinant murine interleukin 4 ( il-4 ) were obtained from jw creagene .
complete freund 's adjuvant ( cfa ) , incomplete freund 's adjuvant ( ifa ) , collagen ( type ii collagen , chicken ) , lps , and mtx were obtained from sigma - aldrich ( st .
cfse ( 5,6-carboxyfluorescein succinimidyl ester ) was obtained from invitrogen ( waltham , ma , usa ) .
recombinant mouse tumor necrosis factor  ( tnf- ) and ccl19 were purchased from r&d systems ( minneapolis , mn , usa ) .
pe - conjugated anti - mouse mhc ii , cd40 , cd11c , ccr7 , and cd80 and fitc - conjugated anti - mouse cd86 , cd54 , and cd14 were purchased from bd pharmingen ( san diego , ca , usa ) .
pe - conjugated anti - mouse foxp3 was purchased from ebioscience ( san diego , ca , usa ) .
dendritic cells were generated from bone marrow progenitors of mice as described previously with some modifications .
briefly , bone marrow cells were prepared from femurs and tibias of mice , and rbcs were lysed with ack lysing buffer ( cmabrex bio science , walkersville , md , usa ) .
cell suspensions were cultured in bacterial plates in the presence of 20  ng / ml of gm - csf and 2  ng / ml of il-4 . at day 3 ,
after 10 days of culture , the semimature and mature dcs were generated by additional incubation with 500  u / ml tnf- and 50  g / ml type ii collagen ( cii ) for 4  h and with 1  g / ml of lps and 50  g / ml cii for 24  h , respectively .
after washing , cells were analyzed by facs caliber ( bd ) using cellquest or flowjo software .
to test dc migration in vitro , the lower chambers of a transwell system ( 8  m pore size , 24 well plate ,
corning , ny , usa ) were filled with ccl19 ( mip3- , 300  ng / ml ) in 0.6  ml and dcs ( 1  10 cells in 0.1  ml ) in serum - free rpmi1640 medium were placed in the upper chambers .
plates were incubated for 3  h at 37c in 5% co2 to allow for migration .
migrated dcs were harvested from the lower chambers , and the total number of cells was determined by cell counting .
dba/1j mice were immunized subcutaneously with 200  g of type ii collagen ( cii , 2  mg / ml ) dissolved in 100  l of 0.05  m acetic acid and mixed with an equal volume ( 100  l ) of cfa . on day 21 , mice were boosted with a subcutaneous injection of cii mixed in ifa .
cia mice were treated with mtx ( 0.1  mg / kg , 0.5  mg / kg , or 1.0  mg / kg ) , vaccinated with cii - pulsed smdc ( 2  10 cells / mouse ) , and/or treated by combination therapy with cii - smdc + mtx ( 0.1 , 0.5 , and 1.0  mg / kg ) . cia mice with an arthritic score of 2 - 3 ( 5 - 6 weeks after primary cii - inoculation ) were treated with two consecutive cycles of mtx intraperitoneally three times a week starting on day 39 and with cii - smdcs injected subcutaneously once a week starting on day 44 .
each therapeutic cycle was completed by administration of mtx on days 2 , 4 , and 6 , followed by administration of cii - smdcs on day 7 .
additionally , cells from spleen and draining lymph nodes were cultured for 72  h in the presence of 50  g / ml of cii and the treg population and ifn-/il-4/il-17/il-10/tgf- secretion from the culture supernatant was determined via facs analysis and elisa .
the degree of arthritis was evaluated from 28 days after the first immunization to up to 8 weeks .
the severity of arthritis was expressed as a mean arthritis index on a 03 scale , as follows : 0 : normal joint ; 1 : slight inflammation and redness ; 2 : severe erythema and swelling affecting the entire paw , with inhibition of use ; and 3 : deformed paw or joint , with ankylosis , joint rigidity , and loss of function . the total score for clinical disease activity was based on all four paws , with a maximum score of 12 for each mouse .
footpad thickness was measured twice a week using a caliper . after sacrificing the mice ,
knee joints were dissected , fixed in 10% phosphate - buffered formalin for 2 days , decalcified in 10% edta for 7 days , dehydrated in an alcohol gradient , and rinsed in running water .
the specimens were processed for paraffin embedding in paraplast ( bdh , dorset , uk ) as routine procedure .
serial paraffin sections measuring 5  m were cut along a longitudinal axis throughout the joint on a microtome , heated at 60c for 30  min , and deparaffinized .
hydration was done by transferring the sections through the following solutions : 70% ethanol , 80% ethanol , 95% ethanol , and 100% ethanol for 2  min twice and xylene for 6  min three times for clarity .
the sections were stained with hematoxylin and eosin ( h&e ) and mounted on glass slides .
splenic t cells were resuspended in pbs ( 0.1% bsa ) at 1  10 cells / ml and incubated with 1  m cfse ( invitrogen ) for 30  min .
dcs ( 1  10 ) were cultured with cfse - labeled spleen t cells ( 1  10 ) for 5 days in 96-well round - bottomed plates ( corning , ny , usa ) .
after 5 days , cells were harvested and the cfse signal of gated lymphocytes was analyzed by flow cytometry .
spleen and lymph nodes were isolated from mice at the peak of clinical cia ( on day 77 ) .
after removal of red blood cells by treatment with 0.16  m tris - nh4cl solution , spleen cell suspensions ( 1  10 cells / ml ) were incubated at 37c for 72  h in complete rpmi 1640 medium containing 50  g / ml cii . after 72  h incubation , the supernatants were collected and ifn- , il-4 , il-17 , il-10 , and tgf- levels were measured using commercially available elisa kits ( quantikine mouse elisa kit , r&d systems , minneapolis , mn , usa ) according to the manufacturer 's protocol . to determine cii - induced cd4foxp3 treg cells , spleen and ln cells
were first stained with fitc - conjugated anti - cd4 antibodies and then fixed and permeabilized with the bd cytofix / cytoperm kit ( bd bioscience pharmingen ) .
cells were then stained with pe - conjugated anti - mouse foxp3 antibody in bd perm / wash buffer for 1  h. after washing with bd perm / wash buffer , cells were analyzed by flow cytometry , in which cd4foxp3 cells were gated from live lymphoid cells . 
 in vitro proliferation of splenocytes
, splenocytes ( 1  10 cells / well in a round bottom , 96-well plate ) were cultured for 5 days at 37c in rpmi medium containing 50  g / ml cii . on the day of the assay , mtt ( 0.5  mg / ml ) was added to the medium in each well , and plates were returned to the incubator for 4  hr .
the absorbance of the samples was then recorded at 570  nm with background subtracted at 630  nm .
the levels of anti - cii antibodies from serum were determined by enzyme - linked immunosorbent assays ( elisas ) as described previously .
briefly , elisa plates ( nunc - immunos , denmark ) were coated with 4  g / ml of cii in 50  mm carbonate buffer at 4c overnight .
fifty microliters of mouse sera diluted at 1/2,000 was added to each well and incubated at rt for 1  hr .
subsequently , the plates were incubated with alkaline phosphatase conjugated goat anti - mouse igg ( southern biotechnology associates , birmingham , al , usa ) and then washed and developed for 520 minutes in a dark room by the addition of p - nitrophenyl phosphate ( sigma ) .
cdna synthesis was performed with the stratascript ii reverse transcriptase ( invitrogen life technologies ) .
pcr was performed using a sybr based assay to examine the level of foxp3 and cytokine mrna with power sybr green pcr master mix ( applied biosystems , carlsbad , ca , usa ) on the steponeplus real - time pcr system ( applied biosystems ) .
samples were in duplicate and relative expression was determined by normalizing to -actin expression in each set of samples to calculate a fold - change value .
arthritogenic t cells collected from the spleen of cia mice at day 21 from first cii injection were cocultured with cii - pulsed smdc at a 10  :  1 ratio for 6 days in the absence or presence of mtx from 0 to 5000  nm .
cells were first stained with fitc - conjugated anti - cd4 antibodies and then fixed and permeabilized with the bd cytofix / cytoperm kit ( bd bioscience pharmingen ) .
intracellular ifn- and foxp3 were then assessed after staining with pe - conjugated anti - mouse ifn- antibody or pe - conjugated anti - mouse foxp3 antibody , respectively , in bd perm / wash buffer for 1  h. after washing with bd perm / wash buffer , cd4ifn- t cells or cd4foxp3 t cells were analyzed by flow cytometry from a live lymphocyte gate .
tnf- modulated smdcs revealed the semimature phenotypes as shown previously , that is , high levels of expression of mhc ii and lower levels of expression of costimulatory molecules such as cd80 , cd86 , and cd40 compared with mdc ( figure 1(a ) ) .
the expression of ccr7 was relatively low in smdcs and increased by maturation ( figure 1(b ) upper ) .
the transwell experiment showed that the mip3-dependent migration capacity of smdcs was similar to the pattern of ccr7 expression ( figure 1(b ) lower ) .
smdcs have much lower t cell proliferation capacity than mdcs in coculture experiments with splenic t cells ( figure 1(c ) ) .
we examined the cytokines produced from t cell / dcs coculture in culture supernatant using elisa .
different from mdcs , smdcs produced relatively high anti - inflammatory cytokines ( immunosuppressive cytokines ) , such as tgf- , il-4 , and il-10 , rather than proinflammatory cytokines such as il-17 and ifn- ( figure 1(d ) ) .
these results suggest that smdcs would be involved in immune regulation rather than immune system activation .
we also found that treg cell population increased when smdcs were incubated with mouse splenocytes ( data not shown ) .
we showed that lower doses of cii - pulsed smdcs ( 2  10 ) can prevent cia when treated before disease onset . in the present study
, we examined the therapeutic effect of cii - pulsed smdcs in advanced cia mice in combination with mtx since most of ra patients are normally treated with mtx until showing mtx - resistance .
when the disease activity score of cia mice reached 2~3 , three different doses of mtx ( oral route , 3 times / week ) and/or smdcs ( subcutaneously , once / week ) were treated for 2 weeks and the ra score was examined . in order to determine the optimal dose of mtx for combination therapy with smdcs , different doses ( from 0.1  mg / kg to 1  mg / kg ) of mtx
were tested in combination with or without smdcs . when mice were treated with smdcs and 0.5  mg / kg or 1  mg / kg mtx ,
treatment with mtx alone did not result in any significant difference in disease progression ( figure 2(a ) ) .
in other words , the severity of arthritis was effectively controlled within the range of arthritic score 3~4 by smdcs alone or by combination therapy for longer than 132 days in cia mice , while the untreated control group reached scores of 7~8 on day 70 and maintained these scores up to day 132 ( figure 2(a ) ) .
joint histopathology data supports the preclinical arthritis score . as shown in the representative h&e staining , both untreated control cia mice and cia mice treated with mtx alone showed significant joint infiltration by leukocytes and severe disruption and loss of articular cartilage ( figure 2(b ) ) .
the cia group treated with smdcs alone or in combination with mtx ( 0.5  mg / kg ) showed nearly intact articular cartilage and subchondral bone and less synovial hyperplasia in the histopathologic analysis ( figure 2(b ) ) .
interestingly , treatment with smdcs alone was also as effective as combination therapy with 0.5  mg / kg mtx in controlling disease progression of cia mice , but the combination of 0.1  mg / kg mtx + smdcs did not show any therapeutic effect ( figures 2(a ) and 2(b ) ) .
these data suggest that ra patients taking mtx can be treated with smdc vaccines without disturbing mtx treatment because mtx is a standard therapy for early ra patients . since it was reported that t cells play an important role in cia onset   and smdcs can induce immune tolerance by t cell hyporesponsiveness , we evaluated cii - specific t cells by proliferation assay with splenic t cells from each group of mice . in comparison with an untreated control group of cia mice ,
cii - specific t cell proliferation was significantly decreased in mice treated with smdcs alone or in combination with 0.5  mg / kg mtx ( figure 3(a ) ) .
these data suggest that the preclinical implications shown in figure 2 would be associated with cii - specific t cell hyporesponsiveness after the treatment of cia mice with smdcs alone or in combination with mtx .
treatment with mtx ( 0.5  mg / kg ) alone also reduced cii - specific t cell proliferation to some amount in cia mice ( figure 3(a ) ) , suggesting that the hyporesponsiveness induced by mtx ( 0.5  mg / kg ) alone would not be enough to control disease progression .
cia mouse serum contains profound amounts of cii - antibody at the time of disease onset .
it has also been suggested that toldcs are involved in the inhibition of b cell - mediated antibody production by blocking t - helper cell function . in vitro
generated smdcs prevent arthritis and inhibit anti - cii antibody production in cia mice [ 25 , 28 ] .
we examined the level of anti - cii antibody in cia mouse serum after treatment with smdcs .
forty days after the onset of ra , anti - cii antibody levels decreased significantly in mice treated with smdcs alone or in combination with mtx , while treatment with mtx alone resulted in no change in serum antibody level ( figure 3(b ) ) , suggesting that there would be a correlation between preclinical outcome and anti - cii antibody level .
levels of proinflammatory cytokines il-17 and tnf- decreased significantly in mice treated with smdcs alone or in combination with mtx when compared to levels in untreated cia mouse sera ( figure 3(c ) ) .
the decrease in tnf- was the most prominent in the 0.5  mg / kg mtx + smdcs combination group , and the serum level of il-1 was significantly reduced when cia mice were treated with mtx alone ( figure 3(c ) ) .
il-1 reduction by mtx treatment was probably due to the mtx - mediated inhibition of il-1 binding to its cell surface receptor . to examine immune status after smdc treatment , we assessed t cell subpopulations in the secondary lymphoid organs of treated mice .
splenic or lymph node ( ln ) t cells were restimulated with cii for 3 days and treg / th17 populations were measured by flow cytometric analysis after intracellular staining or by measuring cytokines in culture supernatants .
untreated cia mice had reduced cd4foxp3 treg population in the spleen and ln compared to the normal control group .
however , the treg populations in both spleen and ln of cia mice significantly increased after treatment with smdcs alone or in combination with mtx ( figure 4(a ) ) .
increase of treg population after the same treatments was further verified by examining foxp3 mrna level in cia mice ( figure 4(a ) lower panel ) . to evaluate the
t cell - mediated immune deviation after these treatments , we examined the t cell subpopulations by measuring the concentration of th1/th2/th17 cytokines in culture supernatants of t cells .
the levels of interferon- ( ifn- , th1 ) and il-17 ( th17 ) decreased significantly in both spleen and ln of cia mice after treatment with smdcs alone or in combination with mtx , while il-4 ( th2 ) was not significantly influenced by the same treatment ( figure 4(b ) ) .
in addition , different from il-4 , immune suppressive il-10 increased significantly in the same treatment ( figure 4(b ) ) .
our findings suggested that the preclinical outcomes shown in figure 2 might be associated with the increase of treg population and decrease of th1/th17 immunity after the therapy with smdcs alone or in combination with mtx .
however , we can not rule out the possibility that other immune responses might be involved in the preclinical outcomes after treatments .
we examined the cytokine milieu and ido expression in secondary lymphoid organs of cia mice after treatment with smdcs alone or in combination with mtx .
the mrna levels of each cytokine and ido were assessed from whole splenocytes and ln cells of cia mice after therapy .
the mrna levels of tnf- , ifn- , and il-17 were significantly reduced in the spleen of cia mice after therapy ( figure 5(a ) ) .
the mrna level of the immunosuppressive cytokine , tgf- , significantly increased in the secondary lymphoid organs after treatment of cia mice with smdcs alone while the level of anti - inflammatory cytokine il-10 mrna was little influenced by treatment ( figure 5(b ) ) .
it is well known that ido is expressed in toldcs and has a role in the induction of treg cells [ 2931 ] .
the mrna level of ido was significantly enhanced only in the ln of cia mice by therapy with smdcs alone , while the level was rather reduced in the spleen after the same treatment ( figure 5(c ) ) .
combination ( smdcs + mtx ) therapy did not result in any significant change in ido level in spleen or ln cells ( figure 5(c ) ) .
even though treatment with mtx alone did not result in a clinical benefit to cia mice ( figure 2 ) , the mtx treatment reduced the level of proinflammatory cytokines tnf- , ifn- , and il-17 in the spleen ( figure 5(a ) ) .
it is worth noting that the reduction of proinflammatory cytokine levels after therapy was more dramatic in the spleen than in the ln , while the increase of immunosuppressive ido and tgf- was more significant in the ln than in the spleen ( figure 5 ) .
we collected splenic t cells from cia mice 21 days after cii - inoculation and cocultured them with smdcs in the presence or absence of mtx in vitro .
ifn- th1 cell population significantly increased among the splenic t cells in cia mice , whereas th17 and il-10 t cells were still at a basal level at this time point in cii - inoculated mice .
coculture with smdcs decreased these ifn- th1 cell population in the splenic t cells regardless of the presence of mtx ( figure 6(a ) ) , suggesting that smdcs were effective in inhibiting inflammatory th1 cells but mtx was not , as was shown in in vivo data ( figure 4(b ) ) . on the other hand , foxp3 treg population increased in cocultures with smdcs or with mtx in a dose dependent manner ( figure 6(b ) ) .
however , smdcs and mtx did not show any synergistic effects on increasing of treg populations ( figure 6(b ) ) .
these results were in good agreement with the in vivo clinical data ( figure 2 ) and the analysis of the splenocytes ( figure 4 ) of cia mice after a combination therapy .
the treatment of ra is based on mtx and other chemical dmards . once dmards do not ameliorate symptoms , biologics , which usually target cytokines , are the next choice . however , biologics have severe side effects in some patients [ 32 , 33 ] and the effect is diminished in some patients by anti - drug antibody formation [ 3436 ] .
most importantly , biologics are not a fundamental treatment , since they generally reduce proinflammatory cytokines and do not remove a fundamental cause of the disease .
tolerogenic dcs ( toldcs ) have been suggested as an attractive solution for fundamental treatment of autoimmune diseases [ 4 , 3739 ] .
the possible mechanism of toldc function in ra therapy is a reduction of th17 population , induction of anergy and apoptosis in effector t cells , suppression of the activation of memory t cells , switching of th1/th2 balance to th2 cells selectively , and induction of regulatory t cells ( treg ) .
. genetic modification of dcs becomes tolerogenic by expression of immunosuppressive genes ( i.e. , il-4 , il-10 , and ctla-4 ) [ 4246 ] or apoptosis - inducing factors ( i.e. , fas , trail ) [ 47 , 48 ] or inhibition of immunostimulatory molecules ( i.e. , cd80/cd86 , il-12 ) [ 49 , 50 ] . immunosuppressive cytokines il-10/tgf- [ 5153 ] or rapamycin treatment , or short - term stimulation with lps , has been used in efforts to make toldcs .
tnf - mediated bmdcs are well known as semimature dcs ( smdcs ) for their surface phenotype , cytokine profile , and biological characteristics in comparison with imdcs and mdcs [ 55 , 56 ] .
we reported that cii - loaded smdcs have a prophylactic effect in cia mice and the preventative effect is dependent on the tolerogenic functions of low - dose smdcs . in the present study ,
we demonstrate that tnf--derived smdcs also have therapeutic potential in advanced cia mice with arthritic scores of 2~3 , and this therapeutic function was still effective when combined with mtx , a first - line agent for ra treatment .
the cia mice treated with cii - smdcs alone or in combination with mtx had a significant reduction in cii - specific cellular and humoral immunities together with proinflammatory cytokines .
foxp3 treg populations were markedly enhanced while th1 and th17 populations decreased significantly with little effect on th2 immunity .
these treatments also increased il-10-expressing t cells in the spleen and induced an anti - inflammatory state in the secondary lymphoid organs of cia mice .
these data suggest that the treatment with cii - smdcs alone or in combination with mtx is effective in controlling disease progression in advanced cia in mice by shifting inflammatory immune environments to a relative anti - inflammatory state . even though mtx alone did produce a clear clinical benefit in advanced cia , treatment of early onset of cia with mtx alone slightly delayed cia progression , probably due to mtx - mediated inhibition of proinflammatory cytokine expression in the spleen . in the mice that received combination therapy with smdcs and mtx ,
the optimal concentration of mtx was 0.5  mg / kg , and low ( 0.1  mg / kg ) and high ( 1.0  mg / kg ) doses of mtx impaired the smdcs - mediated therapeutic efficacy rather than inducing a synergistic effect .
therefore , special attention should be paid to the optimal concentration of mtx in combination therapy with smdcs .
mtx , a standard therapy in ra , is known to affect the immune system [ 1619 ] . to use smdcs in clinic to treat ra in conjunction with mtx
, we have to prove that combination therapy with smdcs and mtx is more effective than mtx alone .
the mtx - only regimen did not have any therapeutic effect in advanced cia . however , the combination of mtx and smdcs was as effective as smdcs alone in the treatment of advanced cia .
our findings suggest that smdcs can be used to treat patients with inadequate response to mtx .
treatment of cia mice with smdcs alone or in combination with mtx was effective in controlling arthritis by decreasing the population of cii - specific th17 and th1 cells in the spleen and increasing the population of treg cells dominant in the ln , leading to inhibition of disease progression .
 we have previously demonstrated that semimature dendritic cell- ( smdc- ) based immunotherapy is effective for the treatment of collagen - induced arthritis ( cia ) prior to disease onset . in the present study 
 , we examined the efficacy of combination therapy with smdcs and methotrexate ( mtx ) in advanced cia with a score of 2 - 3 . 
 combination therapy with low - dose mtx and type ii collagen- ( cii- ) pulsed smdcs ( cii - smdcs ) was more effective in inhibiting disease progression than high or low - dose mtx alone or a combination of high dose mtx and cii - smdcs . 
 the effect of cii - smdcs alone was also comparable to the combination therapy . 
 cd4+foxp3 + treg populations and il-10 secretion markedly increased , and cii - specific autoreactive t cells decreased in mice treated with cii - smdcs alone or in combination with mtx . 
 combination therapy reduced the secretion of interferon- ( ifn- ) and il-17 with little influence on the il-4 secretion in the mixed leukocyte reaction . 
 these results imply that the combination therapy with low - dose mtx and smdcs is effective in controlling advanced cia by enhancing treg population and suppresses antigen - specific th1/th17 immunity , rather than initiating th1 to th2 immune deviation . 
 our findings provide a better understanding of the dc therapy in combination with mtx for the treatment of patients with rheumatoid arthritis ( ra ) . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion