the retroviral plasmid mscv-(sd-)-il-4-neo ( 11 ) contains the poliovirus   type 2 internal ribosome entry site sequence ( 12 ) between the   bacterial neomycin phosphotransferase gene ( neo ) , conferring   neomycin resistance to eukaryotic cells , and the mouse il-4 gene .  
the internal ribosome entry site sequence allows for subsequent   translation of the two proteins .
nolan , manuscript in preparation ) . in brief , pheonix cells were plated at 2    10 cells / well in 6-cm plates and allowed to adhere overnight .
the   cells were transfected with the il-4-neo plasmid ( 10 g / plate ) by   cacl2 transfection .
replication - defective retrovirus was harvested 48 hr after transfection , sterile filtered to remove nonadherent producer cells , and used to infect t cell hybrids .
cells were grown to log phase , harvested ,   and washed two times with pbs .
cells were resuspended to 2    10 cells / ml using retrovirus containing supernatant stocks .
polybrene was added to a final concentration of 8 g / ml . the cells   were placed in 6-well tissue culture plates ( 2 ml / plate ) and centrifuged at 2,500 rpm for 90 min at 32c in a tabletop centrifuge .  
the cells were then incubated for a further 8 h at 37c in a co2   incubator before the cells were washed and replated in fresh media without polybrene . after 48 h of culture to allow gene expression , transduced cells were selected by growth in the presence   of the neomycin analogue g418 ( 2 g / ml ) for 57 d. eae was induced by active immunization with mylein basic protein ( mbp ) . in brief , mbp ( 200 g / mouse final concentration )   in pbs was emulsified in an equal volume of cfa .
sjl/ j)f1 mice ( 5 mice / group ) were immunized subcutaneously at   four sites in the flanks , draining the axial and inguinal lymph   nodes ( 50 l / site ) .
animals were also given an injection of the   coadjuvant pertussis toxin ( 200 ng / mouse ) intravenously on the   day of immunization and 48 h later .
mice were scored daily for   clinical signs of eae according to the following scale : 0 , no clinical disease ; 1 , flaccid tail ; 2 , single hind leg paralysis ; 3 , dual hind   leg paralysis ; 4 , fore limb paralysis ; 5 , moribund ; 6 , death . on day   9 or 10 ,
mice were transferred 10 of each cell type intravenously ,   and were observed daily for clinical signs of eae .
mice were   housed in the stanford department of laboratory animal medicine ( stanford , ca ) under national institutes of health ( nih )   approved conditions . il-4 and
, microtiter plates   were coated with primary antiinterleukin antibody overnight ,   washed with pbs 
after a 2-h incubation , plates were washed and a   biotin - conjugated secondary antiinterleukin antibody was added .  
a standard colormetric assay was then   performed by the addition of the peroxidase substrate 2,2-azinobis ( 3ethylbenzthiazoline-6-sulfonic acid ) .
the retroviral plasmid mscv-(sd-)-il-4-neo ( 11 ) contains the poliovirus   type 2 internal ribosome entry site sequence ( 12 ) between the   bacterial neomycin phosphotransferase gene ( neo ) , conferring   neomycin resistance to eukaryotic cells , and the mouse il-4 gene .  
the internal ribosome entry site sequence allows for subsequent   translation of the two proteins .
nolan , manuscript in preparation ) . in brief , pheonix cells were plated at 2    10 cells / well in 6-cm plates and allowed to adhere overnight .
the   cells were transfected with the il-4-neo plasmid ( 10 g / plate ) by   cacl2 transfection .
replication - defective retrovirus was harvested 48 hr after transfection , sterile filtered to remove nonadherent producer cells , and used to infect t cell hybrids .
cells were grown to log phase , harvested ,   and washed two times with pbs .
cells were resuspended to 2    10 cells / ml using retrovirus containing supernatant stocks .
polybrene was added to a final concentration of 8 g / ml . the cells   were placed in 6-well tissue culture plates ( 2 ml / plate ) and centrifuged at 2,500 rpm for 90 min at 32c in a tabletop centrifuge .  
the cells were then incubated for a further 8 h at 37c in a co2   incubator before the cells were washed and replated in fresh media without polybrene . after 48 h of culture to allow gene expression ,
transduced cells were selected by growth in the presence   of the neomycin analogue g418 ( 2 g / ml ) for 57 d.
eae was induced by active immunization with mylein basic protein ( mbp ) . in brief , mbp ( 200 g / mouse final concentration )   in pbs was emulsified in an equal volume of cfa .
sjl/ j)f1 mice ( 5 mice / group ) were immunized subcutaneously at   four sites in the flanks , draining the axial and inguinal lymph   nodes ( 50 l / site ) .
animals were also given an injection of the   coadjuvant pertussis toxin ( 200 ng / mouse ) intravenously on the   day of immunization and 48 h later .
mice were scored daily for   clinical signs of eae according to the following scale : 0 , no clinical disease ; 1 , flaccid tail ; 2 , single hind leg paralysis ; 3 , dual hind   leg paralysis ; 4 , fore limb paralysis ; 5 , moribund ; 6 , death . on day   9 or 10 ,
mice were transferred 10 of each cell type intravenously ,   and were observed daily for clinical signs of eae .
mice were   housed in the stanford department of laboratory animal medicine ( stanford , ca ) under national institutes of health ( nih )   approved conditions .
, microtiter plates   were coated with primary antiinterleukin antibody overnight ,   washed with pbs 
an   il-4 or il-10 standard was included in each assay for quantification purposes . after a 2-h incubation
, plates were washed and a   biotin - conjugated secondary antiinterleukin antibody was added .  
a standard colormetric assay was then   performed by the addition of the peroxidase substrate 2,2-azinobis ( 3ethylbenzthiazoline-6-sulfonic acid ) .
to test the concept of site - specific delivery of cytokines   by transduced t cells , the murine model of eae was used .
an   encephalitogenic , mbp - specific t cell hybridoma , g1.15h ,   was used in these preliminary studies due to its transduction   efficiency .
 pl / j)f1 mice verified its eae - inducing ,   and thus cns - homing , capability ( fig .
it was decided   to transduce this t cell line for expression of the il-4 gene   since this cytokine has been demonstrated to be a mediator   in eae regulation as evidenced by its role in the natural recovery from disease ( 13 ) , experimental protection from disease after oral tolerance induction ( 14 ) , and after immunization with altered peptide ligands of mbp ( 15 ) .  
after transduction of g1.15h with an il-4encoding   retrovirus and drug selection , the line was confirmed to secrete il-4 by elisa ( table 1 ) .
subsequent limiting dilution cloning of these transduced hybrids yielded individual   lines secreting between 1 and 8 pg / ml il-4/10 cells ( table 1 ) .  
10 d after mbp immunization to induce eae , ( sjl / j    pl / j)f1 mice were given transduced or control cells .
clinical eae was ameliorated by adoptive transfer of 10 transduced t cells secreting high levels of il-4 ( line n5 ) .
disease onset for the il-4 treatment group was delayed by 2 d   ( p < 0.01 student 's t test ) and the average disease score was   significantly lower ( p < 0.05 ) than an experimental group   receiving cells transduced to express il-10 , or control mice   receiving untransduced cells or pbs ( fig .
that amelioration of disease was due to local delivery of   il-4 was supported by several experimental approaches . in   the first , mice that received t cells transduced to express   il-4 were bled at various time points after cell transfer , and   serum il-4 was determined by elisa .
no cytokine was   detected in the serum until day 24 after transfer , well after   initial recovery from disease .
serum il-4 levels at this time   were low , ranging from 1.19 pg / ml to 2.53 pg / ml ( table   2 ) .
this finding indicates that disease remission was not due   to high systemic levels of il-4 . in a second experimental   approach
, we verified the presence of transduced t cells in   the cns at the time of disease amelioration by testing spinal cord tissue from treated animals for retroviral - specific   il-4 expression by reverse transcriptase pcr analysis .
retroviral il-4 transcription could be detected in the cns of   treated animals 15 d after transfer of transduced t cells ( data   not shown ) .
the third experimental approach demonstrated that amelioration of disease was dependent on t cell   homing to the cns .  
we reasoned that t cells transduced to express il-4 that   could not recognize cns antigens would be ineffective at   delivering il-4 to the cns . to test this hypothesis , additional transfer experiments were performed , using as controls , transduced t cells expressing il-4 but lacking antigen   specific tcr expression .
in the first experiment , an il-4 expressing transductant of the hybridoma fusion partner   bw5147 was used as a control .
transfer of this cell line to   mbp - immunized mice had no effect on the disease course ,   whereas transfer of an mbp - specific line , clone 4.9 , secreting low levels of il-4 ( matched to the fusion partner control ) had a significant therapeutic effect ( p < 0.05 ; data not   presented ) .
in the second experimental approach , a tcr   negative variant of the il-4secreting disease ameliorating   n5 clone was used as a control . this line had significantly   less effect on the disease course ( p < 0.05 ) when compared   to its tcr - expressing counterpart ( fig .
3 ) .   the majority of approaches taken to control autoimmune disease result in deleterious side effects due to the   systemic administration of antiinflammatory agents .
our   findings indicate that the disease processes can be modulated by the transfer of mbp - specific t cells which have   been retrovirally modified to express the antiinflammatory   cytokine il-4 .
disease amelioration was due to local , rather   than systemic , delivery of il-4 as evidenced by the following points .
( a ) serum levels of il-4 were undetectable at   the time of disease remission .
( b ) retroviral - encoded il-4   expression could be detected in the cns of treated mice .  
( c ) disease amelioration was dependent upon antigen - specific t cell receptor expression on transduced t cells , indicating that t cell antigen recognition and presumably  trafficking  were necessary for delivery of cytokines .
the use of antigen - specific t cells , transduced to express   regulatory cytokines , selectively target antiinflammatory   molecules to the site of pathology represents a unique therapeutic approach to the treatment of autoimmune disease .  
t cells are advantageous since they are easily manipulated   and expanded in tissue culture before reintroduction into   the host .
more importantly , the antigen specificity of t   cells allows them to home to depots of antigen in the body ,   such as at inflammatory sites of autoimmune disease .
this   has been demonstrated in the murine model of eae where   mbp - specific t cells have been shown to traffic to the   cns , both during the induction phase of disease as well as   during relapses in a relapsing - remitting model of eae ( 16 ,   17 ) .
results presented here demonstrate that a statistically   significant benefit can be observed when mice , immunized   to develop eae , are given mbp - specific t cells retrovirally   transduced to express il-4 .
three naive ( pl / j  sjl / j)f1 mice were given 10   g1.15h cells intravenously on day 0 . each mouse ( circles , squares , or triangles , respectively ) was scored daily for clinical signs of eae as described in   materials and methods . il-4 and il-10 secreted by cell lines used in this study control il-4 and il-10transduced cell lines were incubated overnight , and il-4 and il-10 in culture supernatants quantitated by elisa as described in materials and methods
.        adoptive transfer of t cells transduced to express il-4 ameliorates mbp - induced eae .
mice immunized to induce eae were   treated with t cells transduced to express il-4 ( circles ) or il-10 ( triangles ) ,   untransduced t cells ( diamonds ) , or pbs ( squares ) , and observed for clinical   signs of eae .
data are given as mean disease scores , and are representative   of the results of at least two independent experiments .
the mean disease score was statistically different   from all other groups , student 's t test , p < 0.05 .
serum levels of il-4 at various times after transfer of t cell hybrids transduced to express il-4 n5-transferred mice were bled from the tail vein on the indicated days   into heparinized tubes .
serum was obtained by centrifugation and tested   for il-4 by elisa as described in materials and methods .
none   detected ( lower limit of detection of elisa = 0.875 pg / ml ) .       
mbp - immunized ( pl / j  sjl / j)f1 mice ( 10   mice / group ) were given 10 transduced tcr - positive , il-4secreting   n5 cells ( circles ) or tcr - negative , il-4secreting n5 cells ( squares ) .
data are given as the mean   disease scores of those mice developing disease , and are representative of   the results of at least two independent experiments .
the mean disease score was statistically different   from all other groups , student 's t test , p < 0.05 .
 experimental autoimmune encephalomyelitis ( eae ) is an inflammatory autoimmune disease of   the central nervous system which serves as a model for the human disease multiple sclerosis .   
 we demonstrate here that encephalitogenic t cells , transduced with a retroviral gene , construct to express interleukin 4 , and can delay the onset and reduce the severity of eae when   adoptively transferred to myelin basic protein  immunized mice . 
 thus , t lymphocytes transduced with retroviral vectors can deliver  regulatory cytokines  in a site - specific manner and   may represent a viable therapeutic strategy for the treatment of autoimmune disease . 
Materials and Methods
Retroviral Constructs and Ecotropic Viral Production.
T Cell Infections.
Induction of EAE and Adoptive Transfer of Transduced T Cells.
Quantitation of IL-4 and IL-10 by ELISA.
Results and Discussion
Figures and Tables