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Paper text:
7 m acetone - fixed frozen sections from spleen or kidney were air - dried and stained with various antibodies for 30 min at room temperature in a moist chamber . for the identification of germinal centers , sections were labeled with pe conjugated anti - b220 antibodies ( bd , ra3 - 6b2 ) and fitc conjugated anti - gl-7 ( bd , clone gl7 ) antibodies . for assessment of igg deposition in the kidney ,
> 10 sections stained with fitc - conjugated anti - mouse igg ab ( sigma ) were examined for each experimental condition , verified for reproducibility and quantified as positively - stained using imagej software .
single - cell suspensions were prepared and maintained in the dark at 4c for immunofluorescent analysis , washed in ice - cold facs buffer ( 2% fatal calf serum , 0.1% nan3 in pbs ) and incubated with each antibody for 30 min and washed with facs buffer before analyzing .
anti - cd4 , anti - cd8 , anti - b220 , anti - cd44 , anti - cd62l , anti - fas , anti - qa-1b , anti - cd45.2 , anti - pd-1 , anti - cd11b , anti - cxcr5 , anti - ifn , anti - il10 , anti - il4 , anti - icos , anti - vb5.1/5.2 , anti - nkg2a , anti - igm ( bd bioscience ) or anti - cd8 , anti - ctla4 , anti - ccr7 , anti - gitr , anti - icosl , anti - cd127 , anti - cd25 , anti - klrg1 and anti - vla4 ( ebioscience ) were used followed by analysis of cells using a facscalibur ( bd biosciences ) and flowjo software ( tristar ) . for detection of np specific antibodies ,
elisa plates were coated with 0.5 g / ml np4-bsa or 1 g / ml np23-bsa ( biosearch technologies ) and serum harvested 14 days after immunization with np19-klh in cfa and reimmunization with np19-klh in ifa was used as a standard .
total igg , igg1 and igg2a were detected by incubating plates with biotinylated anti - mouse igg , igg1 or igg2a followed by streptavidin - peroxidase .
anti - dsdna and anti - nuclear antibodies in mouse sera were determined by elisa ( alpha diagnostic international ) and porcine thyroglobulin ( sigma ) and porcine insulin ( sigma ) were used to detect relevant ab .
ifn and il-4 were measured using opt elisa kit ( bd biosciences ) ; il-21 and il-17 were measured using elisa duo set ( r&d systems ) .
68 week old or 6 month old b6.qa-1(wt ) or b6.qa-1(d227k ) mice were infected i.p . with 110 pfu lcmv - armstrong .
at day 8 , mice were reinfected i.p . with 110 pfu lcmv - armstrong .
serum samples were prepared at day 8 ( prior to reinfection ) and day 13 and subjected to the elisa test ( biotech trading partners ) .
cells from spleen and lymph nodes were collected from wt ot - ii or b6.qa-1(d227k ) ot - ii mice .
210 cells / ml ot - ii cd4 cells were stimulated with 1 g / ml ot - ii peptides ( isqavhaahaeineagr ) in the presence of 210 irradiated total splenocytes .
for the differentiation of ot - ii cd4 cells to follicular helper t cell phenotype , 50 ng / ml il-21 , 10 g / ml anti - ifn ab , 10 g / ml anti - il-4 ab , 20 g / ml ril-6 , and 20 g / ml anti - tgf ( 1d11 ) abs were added into the culture . at day 5 ,
recipient mice were immunized i.p . with either 100 g np13-ova in cfa or 100 g np19-klh in cfa .
statistical analyses were performed using wilcoxon - mann - whitney rank sum test for comparison of two conditions and kruskal - wallis test for comparison of more than two conditions .
p value < 0.05 was considered statistically significant ( * = < 0.05 , * * = < 0.01 , * * * = < 0.001 ) .
7 m acetone - fixed frozen sections from spleen or kidney were air - dried and stained with various antibodies for 30 min at room temperature in a moist chamber . for the identification of germinal centers , sections were labeled with pe conjugated anti - b220 antibodies ( bd , ra3 - 6b2 ) and fitc conjugated anti - gl-7 ( bd , clone gl7 ) antibodies . for assessment of igg deposition in the kidney ,
> 10 sections stained with fitc - conjugated anti - mouse igg ab ( sigma ) were examined for each experimental condition , verified for reproducibility and quantified as positively - stained using imagej software .
single - cell suspensions were prepared and maintained in the dark at 4c for immunofluorescent analysis , washed in ice - cold facs buffer ( 2% fatal calf serum , 0.1% nan3 in pbs ) and incubated with each antibody for 30 min and washed with facs buffer before analyzing .
anti - cd4 , anti - cd8 , anti - b220 , anti - cd44 , anti - cd62l , anti - fas , anti - qa-1b , anti - cd45.2 , anti - pd-1 , anti - cd11b , anti - cxcr5 , anti - ifn , anti - il10 , anti - il4 , anti - icos , anti - vb5.1/5.2 , anti - nkg2a , anti - igm ( bd bioscience ) or anti - cd8 , anti - ctla4 , anti - ccr7 , anti - gitr , anti - icosl , anti - cd127 , anti - cd25 , anti - klrg1 and anti - vla4 ( ebioscience ) were used followed by analysis of cells using a facscalibur ( bd biosciences ) and flowjo software ( tristar ) .
for detection of np specific antibodies , elisa plates were coated with 0.5 g / ml np4-bsa or 1 g / ml np23-bsa ( biosearch technologies ) and serum harvested 14 days after immunization with np19-klh in cfa and reimmunization with np19-klh in ifa was used as a standard .
total igg , igg1 and igg2a were detected by incubating plates with biotinylated anti - mouse igg , igg1 or igg2a followed by streptavidin - peroxidase .
anti - dsdna and anti - nuclear antibodies in mouse sera were determined by elisa ( alpha diagnostic international ) and porcine thyroglobulin ( sigma ) and porcine insulin ( sigma ) were used to detect relevant ab .
ifn and il-4 were measured using opt elisa kit ( bd biosciences ) ; il-21 and il-17 were measured using elisa duo set ( r&d systems ) .
68 week old or 6 month old b6.qa-1(wt ) or b6.qa-1(d227k ) mice were infected i.p . with 110 pfu lcmv - armstrong . at day 8
serum samples were prepared at day 8 ( prior to reinfection ) and day 13 and subjected to the elisa test ( biotech trading partners ) .
cells from spleen and lymph nodes were collected from wt ot - ii or b6.qa-1(d227k ) ot - ii mice .
210 cells / ml ot - ii cd4 cells were stimulated with 1 g / ml ot - ii peptides ( isqavhaahaeineagr ) in the presence of 210 irradiated total splenocytes .
for the differentiation of ot - ii cd4 cells to follicular helper t cell phenotype , 50 ng / ml il-21 , 10 g / ml anti - ifn ab , 10 g / ml anti - il-4 ab , 20 g / ml ril-6 , and 20 g / ml anti - tgf ( 1d11 ) abs were added into the culture . at day 5 , live cd4 cells were harvested from the culture by percoll gradient centrifugation . ot -
recipient mice were immunized i.p . with either 100 g np13-ova in cfa or 100 g np19-klh in cfa .
statistical analyses were performed using wilcoxon - mann - whitney rank sum test for comparison of two conditions and kruskal - wallis test for comparison of more than two conditions .
p value < 0.05 was considered statistically significant ( * = < 0.05 , * * = < 0.01 , * * * = < 0.001 ) .
the ability to produce vigorous immune responses that spare self tissues and organs depends on elimination of autoreactive t and b cells . 
 however , purging of immature and mature self - reactive t and b cells is incomplete and may require additional censorship by cells programmed to suppress immune responses 1 . 
 regulatory t cells belonging to the cd4 + t cell subset may play a role in preventing untoward inflammatory responses , but t cell subsets programmed to inhibit the development of autoantibody formation and sle - like disease have not been defined 2 . 
 here we delineate a cd8 + regulatory t cell lineage that is essential for maintenance of self tolerance and prevention of autoimmune disease . 
 genetic disruption of the inhibitory interaction between these cd44 + icosl+ cd8 + t cells and their target qa-1 + follicular t helper cells results in the development of a lethal sle - like autoimmune disease . 
 these findings define a sublineage of cd8 t cells programmed to suppress rather than activate immunity that represents an essential regulatory element of the immune response and a guarantor of self tolerance .