female nod / ltj and bdc2.5-nod , wild - type c57bl/6 , h-2 c57bl/6 , and mhc ii c57bl/6 were purchased from the jackson laboratory ( bar harbor , me ) .
all mice were maintained in a pathogen - free room at city of hope research animal facilities ( duarte , ca ) .
flow cytometry staining and analysis , cd11c enrichment , and cd4 t - cell depletion were performed as described ( 3335 ) .
phycoerythrin ( pe)labeled bdc2.5-mimotope tetramer ( i - a ahhpiwarmda ) and control - tetramer ( i - a pvskmrmatpllmqa ) were obtained from nih tetramer facility ( atlanta , ga ) .
analysis of insulitis and -cell surface area was performed as previously described ( 33 ) .
comparison of t1d development was evaluated using the log - rank test , and comparison of means was evaluated using unpaired two - tailed student t tests with prism version 5 ( graphpad , san diego , ca ) .
flow cytometry staining and analysis , cd11c enrichment , and cd4 t - cell depletion were performed as described ( 3335 ) .
phycoerythrin ( pe)labeled bdc2.5-mimotope tetramer ( i - a ahhpiwarmda ) and control - tetramer ( i - a pvskmrmatpllmqa ) were obtained from nih tetramer facility ( atlanta , ga ) .
analysis of insulitis and -cell surface area was performed as previously described ( 33 ) .
comparison of t1d development was evaluated using the log - rank test , and comparison of means was evaluated using unpaired two - tailed student t tests with prism version 5 ( graphpad , san diego , ca ) .
it was reported that induction of chimerism with bm cells from mhc - mismatched nonautoimmune donors prevented t1d development ( 28,3034 ) .
however , it was not clear whether those chimeric recipients had mixed or complete chimerism , because in those reports chimerism was measured with peripheral blood mononuclear cells , and the mixed chimerism could consist of residual host - type t - cells without de novo developed host - type t - cells . to identify the true mixed chimerism , de novo developed donor- and host - type cd4cd8 thymocytes , as well as b220igm or mac1/gr1 bm resident cells , needed to be measured . therefore ,
we used a recently reported radiation - free and graft versus host disease ( gvhd ) preventative anti - cd3/cd8-based condition regimen ( 3336 ) to induce mixed or complete chimerism by transplanting graded numbers of donor cells . accordingly ,
6-week - old nod mice ( h-2k , i - a , cd45.1 ) were injected with anti - cd3 and anti - cd8 ( 5 g / g each ) on days 10 and 5 . on day 0 , the conditioned mice were intravenously injected with graded numbers ( 550  10 ) of bm and cd4 t - depleted spleen cells from mhc - mismatched c57bl/6 ( h-2k , i - a , cd45.2 ) donors .
the recipients were checked for chimerism with blood mononuclear cells and monitored weekly for t1d development for up to 100 days after hct .
thereafter , the pancreas , spleen , bm , and thymus of the recipients and control mice were harvested for evaluation of insulitis and confirmation of chimerism status . because we have reported that blood chimerism levels become stable in nod recipients conditioned with the radiation - free anti - cd3-based regimen 60 days after hct ( 34 ) , and this notion
was confirmed with representative recipients in the current study ( supplementary fig . 1 and supplementary table 1 )
we found that the development of mixed and complete chimerism was associated with donor cell dose .
in general , recipients given > 25  10 mhc - mismatched donor cells all developed complete chimerism , and the majority of recipients given < 20  10 donor cells developed mixed chimerism as judged by analysis of donor- and host - type t - cells , b - cells , and macrophages / granulocytes among blood mononuclear cells .
we also found that , 100 days after hct , 66% ( 8 of 12 ) of the control mice , but none of the mixed or complete chimeric recipients , developed t1d ( p < 0.01 , fig . 1a ) .
although almost all of the residual islets in control mice had severe insulitis , none of the islets in the mixed and complete chimeric recipients had insulitis ( p < 0.01 , fig .
1b ) , although a small portion of them showed very minor peri - insulitis ( fig .
1b ) and there was no significant difference between mixed and complete chimeric recipients ( p > 0.5 , fig .
2 . mixed and complete chimerism with mhc - mismatched bm transplants both prevented t1d in nod mice .
wild - type nod mice were conditioned with anti - cd3/cd8 ( 5 g each ) on days 10 and 5 . on day 0
, the conditioned mice were transplanted with graded doses of cd4 tcd splenocytes and whole bm from wild - type c57bl/6 donors to induce chimerism ( < 20  10 each for the mixed , 50  10 each for the complete chimeras ) .
diabetes development was monitored weekly by both urine and blood glucose for up to 100 days .
thereafter , the recipient pancreas , spleen , bm , and thymus were harvested for evaluation of insulitis and chimerism status .
c : one representative spleen cell facs profile of eight recipients examined with anti - h-2k in each group . for dc patterns ,
d : one representative facs profile of four recipients in each group examined with anti - cd45.2 and anti - cd45.1 for donor- or host - type b220igm or mac1/gr1 cells in the bm .
e : representative facs profile of four recipients in each group examined with anti - h-2k and anti - h-2k for donor- and host - type thymocytes .
furthermore , we confirmed the status of chimerism by examining the spleen , bm , and thymus of the recipients .
we observed that , in the mixed chimeras , more than one - third of the t - cells , b - cells , macrophages , and granulocytes in the spleen were host - type .
in contrast , in the complete chimeras , less than 1% of the t- and b - cells were host - type , although 20% of the macrophage and granulocytes were host - type ( fig .
we should point out that those h-2 cells may not be true host - type cells , because we found that 10% of the mac-1/gr-1 cells in the spleen of c57bl/6 mice are h-2 ( supplementary fig . 3 and supplementary table 2 ) .
the mixed chimeric recipients had both donor- and host - type b220igm immature b - cells and mac1gr1 myeloid cells in the bm , whereas the complete chimeras had only donor - type cells ( fig .
consistently , the mixed chimeric recipients had both donor- and host - type cd4cd8 thymocytes , and the complete chimeric recipients had only donor - type but no host - type thymocytes ( fig .
furthermore , after enrichment of cd11c cells from thymus , we observed donor cd11c dendritic cells ( dcs ) in the thymus of both mixed and complete chimeric recipients ( fig .
this result is consistent with a previous report showing that donor - type dcs engraft in the thymus in the chimeric recipients as judged by immunohistochemistry ( ihc ) staining ( 37 ) .
percent donor- and host - type cells in spleen of mixed and complete chimeras given mhc - mismatched bm transplants data are means  se ; n = 4 .
donor - type cells were defined as h-2k , and host - type cells were defined as h-2k . taken together , these results indicate that induction of mixed chimerism with mhc - mismatched bm transplants is as efficient as induction of complete chimerism in prevention of insulitis and t1d .
( 29 ) that induction of mixed chimerism in lethally irradiated nod recipients with bm cells from mhc - matched h-2 donors prevents t1d , but it was not clear whether those chimeric recipients were true mixed chimeras with de novo developed host - type t - cells . to clarify this issue , we performed a similar experiment by transplanting h-2 c57bl/6 ( h-2k , i - a , cd45.2 ) donor t - cell depleted ( tcd ) bm cells ( 10  10 ) into lethally irradiated nod ( h-2k , i - a , cd45.1 ) recipients .
in addition , we induced chimerism in anti - cd3/cd8-conditioned nod recipients by transplanting bm and cd4 t - depleted spleen cells ( 50  10 each ) from h-2 c57bl/6 donors . the control nod mice were provided anti - cd3/cd8 conditioning only .
the recipients and control mice were monitored for t1d development and checked for chimerism as described above .
( 29 ) , the total body irradiation ( tbi)-conditioned nod recipients given tcd bm cells from mhc - matched h-2g7 c57bl/6 donors appeared to have so called mixed chimerism , as judged by the presence of both donor- and host - type t- and b - cells among blood mononuclear cells ( data not shown ) , and no recipients developed t1d by 100 days after hct , although 61% ( 11/18 ) of control nod given conditioning only developed t1d ( p < 0.01 , fig . 2a ) .
the anti - cd3/cd8-conditioned nod recipients from bm and cd4 t - depleted spleen cells also showed mixed chimerism among blood mononuclear cells ( data not shown ) , but 17% ( 2/12 ) of them developed t1d by 100 days after hct , although there was still a significant difference compared with nod mice given conditioning only ( p < 0.05 , fig .
2a ) . to further analyze the difference between the tbi - conditioned and the anti - cd3/cd8-conditioned chimeras , the pancreata of the chimeric recipients and control mice were evaluated for insulitis and their spleen , bm , and thymus were measured for chimerism .
we found that , although the tbi - conditioned chimeric animals had little insulitis , the anti - cd3/cd8-conditioned chimeras had severe insulitis ( p < 0.01 , fig . 2b ) and marked reduction of -cell surface as compared with the tbi - conditioned recipients ( p < 0.01 , fig . 2c ) .
these results indicate that insulitis is eliminated in tbi - conditioned but not in anti - cd3-conditioned chimeras .
wild - type nod mice were conditioned with anti - cd3/cd8 on days 10 and 5 . on day 0 , the conditioned mice were transplanted with cd4-tcd splenocytes and bm ( 50  10 each ) from mhc - matched h2- c57bl/6 mice to induce mixed chimerism . to induce mixed chimerism with tbi conditioning , wild - type nod
diabetes development was monitored weekly by both urine and blood glucose for up to 100 days after hct .
thereafter , the recipient pancreas , spleen , bm , and thymus were harvested for evaluation of insulitis and chimerism status . a : diabetes development curve after hct ( n = 12 for anti - cd3-conditioned chimeric recipients ; n = 18 for conditioning alone : n = 6 for tbi - conditioned chimeric recipients ) .
b : percent insulitis ( n = 4 for anti - cd3-conditioned chimeric recipients ; n = 6 for tbi - conditioned chimeric recipients and conditioning alone ) .
c : percent -cell surface area as compared with total pancreatic tissue surface area ( n = 46 ) .
d : representative facs profiles showing spleen chimerism pattern 100 days after hct ( n = 12 ) .
for dc patterns , spleens were digested with collagenase d and enriched using cd11c microbeads .
e : representative facs profiles showing donor- or host - type b220igm or mac1/gr1 cells in the bm ( n = 4 ) .
furthermore , we found that , although there were both donor- and host - type t - cells , b - cells , and macrophage / granulocytes among the spleen cells of tbi - conditioned or anti - cd3/cd8-conditioned chimeras , the donor - type cells were dominant in the former , which was consistent with the report of beilhack et al .
( 29 ) , but the host - type cells were dominant in the latter ( fig .
in addition the immature b220igm b - cells and mac-1/gr-1 myeloid cells in the bm were almost all donor - type in the tbi - conditioned recipients , although there were both donor- and host - type cells in the anti - cd3/cd8-conditioned chimeras ( fig .
consistently , although there were both donor- and host - type thymocytes in the tbi - conditioned chimeras , there were only donor - type but no host - type cd4cd8 thymocytes in the tbi - conditioned chimeras .
in contrast , both donor- and host - type cd4cd8 thymocytes were present among the thymocytes of anti - cd3/cd8-conditioned chimeras ( fig .
finally , there was only donor - type cd11c dcs in the thymus of tbi - conditioned chimeras , although there were both donor- and host - type cd11c dcs in the anti - cd3/cd8 conditioned chimeras ( fig .
percent donor- and host - type cells in spleen of chimeras given mhc - matched bm transplants data are means  se ; n = 4 .
donor - type cells were defined as cd45.2 , and host - type cells were defined as cd45.2 . taken together , the so - called mixed chimerism in lethally irradiated tbi - conditioned chimeras consists of radiation - resistant residual host - type but no de novo developed host - type hematological cells , and they are actually complete chimeras . in contrast , the mixed chimerism in anti - cd3/cd8-conditioned recipients consists of both residual and de novo developed host - type hematological cells , and they are truly mixed chimeras .
these results indicate that induction of mixed chimerism with mhc - matched donor bm transplants can not prevent insulitis , although induction of complete chimerism can .
we next tested whether mhc - mismatched but not mhc - matched bm transplants were able to restore thymic negative selection of autoreactive t - cells in the mixed chimeric nod mice , using t - cell receptor ( tcr ) transgenic bdc2.5-nod mice ( 38 ) .
in addition , we tested whether donor cell expression of mismatched mhc ii molecules was also required . accordingly , anti - cd3-conditioned bdc2.5-nod ( h-2k , i - a , cd45.1 ) mice were induced to develop mixed chimerism by injecting whole bm cells ( 50  10 ) from mhc - mismatched wild - type or mhc ii c57bl/6 ( h-2k , i - a , cd45.2 ) or mhc - matched h-2 c57bl/6 ( h-2k , i - a , cd45.2 ) donors .
the chimerism was evaluated by fluorescence - activated cell sorting ( facs ) analysis of blood mononuclear cells as described above ( data not shown ) , and the recipients were monitored for t1d development .
we found that although 75% ( 9/12 ) of control bdc2.5-nod mice given anti - cd3 conditioning only developed t1d , none ( 0/12 ) of the mixed chimeric recipients with mhc - mismatched bm showed t1d 160 days after hct ( p < 0.01 , fig . 3a ) .
in addition , although the control mice had severe insulitis , the mixed chimeras with mhc - mismatched wild - type bm showed no insulitis ( p < 0.01 ) , although they showed mild peri - insulitis ( fig .
it is of interest that induction of mixed chimerism with mhc - mismatched mhc ii or mhc - matched h-2 donor bm cells not only failed to prevent but markedly augmented t1d development , as compared with the control bdc2.5-nod mice ( p < 0.01 , fig . 3a ) . the mixed chimerism status in the different group was confirmed by flow cytometry analysis of the recipient spleen cells ( fig .
these results indicate that induction of mixed chimerism with mhc - mismatched , but not mhc - matched , donor bm cells can prevent t1d development in transgenic autoimmune bdc2.5-nod mice , and the expression of mhc ii molecules by the mhc - mismatched donor cells is required for disease prevention .
mixed chimerism with mhc - mismatched wild - type bm prevented t1d in transgenic bdc2.5-nod mice .
transgenic bdc2.5-nod mice were conditioned anti - cd3 ( 5 g ) on day 7 . on day 0 , the conditioned mice were transplanted with bm cells ( 50  10 ) from mhc - mismatched wild - type , mhc - mismatched mhc ii or mhc - matched h-2 c57bl/6 donors to induce mixed chimerism .
diabetes development was monitored weekly by both urine and blood glucose . a : diabetes development curve after hct ( n = 12 for mice given conditioning alone or recipients given bm cells from mhc - mismatched wild - type donors ; n = 7 for recipients given mhc - mismatched mhc ii donor bm cells ; n = 8 for recipients given mhc - matched bm cells ) .
b : percent insulitis for recipients given mhc mismatched wild - type bm versus conditioning alone 120 days after transplantation ( n = 4 ) .
we next tested whether there was a deletion of de novo developed host - type autoreactive t - cells in the thymus of the mixed chimeras given mhc - mismatched wild - type bm cells .
we first compared the percentage of donor- and host - type thymocytes at cd4cd8 , cd4cd8 , and cd4cd8 stages ( fig .
bdc2.5-nod mice are a cd4 t transgenic mouse line ( 38 ) , thus , we did not evaluate cd4cd8 thymocytes .
we found that host - type thymocytes were present among cd4cd8 , cd4cd8 , and cd4cd8 in anti - cd3-conditioned control bdc2.5-nod mice without hct as well as in the mixed chimeras given mhc - mismatched mhc ii or mhc matched h-2 donor bm cells .
in contrast , the host - type thymocytes in the mixed chimeras given mhc - mismatched donor bm cells were mainly present among cd4cd8 and cd4cd8 thymocytes , and they were nearly undetectable among total cd4cd8 thymocytes ( fig .
after cd45.1 host - type cells were gated on , a small percentage of cd4cd8 cells were detected in those mixed chimeric recipients , although it was 15-fold lower , as compared with control bdc2.5 mice or the mixed chimeras given mhc - mismatched mhc ii or mhc - matched donor bm cells ( p < 0.01 , fig .
interestingly , the reduction of the percentage of cd4cd8 subset among host - type thymocytes in the mixed chimeras given mhc - mismatched wild - type donor bm cells was associated a more than 20-fold increase of cd4cd8 subsets ( p < 0.01 , fig .
these results indicate that host - type cd4cd8 thymocytes in the mixed chimeras given mhc - mismatched donor bm cells are prevented to develop into cd4cd8 stage .
mixed chimerism with mhc - mismatched wild - type bm transplants mediated thymic deletion of de novo developed autoreactive t - cells .
transgenic bdc2.5-nod mice were conditioned anti - cd3 ( 5 g ) on day 7 . on day 0 , the conditioned mice were transplanted with bm cells ( 50  10 ) to induce mixed chimerism .
mice were analyzed for clonal deletion of autoreactive t - cells at time of diabetes development , 3060 days after hct .
a : representative facs profiles showing clonal deletion of autoreactive t - cells in the thymus .
cd45.1 host - type thymocytes were gated on and shown in cd4 versus cd8 , and cd4cd8 cells were then gated and shown in histogram of bdc2.5-tetramer ( solid line ) versus control - tetramer ( shaded area ) .
host - type cd45.1cd4 splenocytes were gated on and shown in histogram of bdc2.5-tetramer ( solid line ) versus control - tetramer ( shaded area ) ( n = 4 ) .
c  f : yield of host - type tetramer cells among cd4cd8 , cd4cd8 , and cd4cd8 thymocytes as well as cd4 splenocytes ( n = 4 ) .
deletion of transgenic autoreactive thymocytes has been reported to take place at cd4cd8 stage ( 39,40 ) .
thus we tested whether this is the case in the mixed chimeric bdc2.5-nod recipients , using i - a - mimotope - tetramer ( bdc2.5-tetramer ) to identify the autoreactive bdc2.5 t - cells , as previously described ( 41 ) .
we found that 50% of the host - type cd4cd8 thymocytes in the control bdc2.5 mice given anti - cd3-conditioning only and the mixed chimeras given mismatched mhc ii or mhc - matched donor bm cells were bdc2.5-tetramer and that there was no significant difference among the three groups ( fig .
, the bdc2.5-tetramer cells among the host - type cd4cd8 thymocytes in the mixed chimeras given mhc - mismatched wild - type donor bm cells were reduced by approximately fivefold as compared with control bdc2.5-nod mice ( p < 0.01 , fig .
the yield of cd4cd8 bdc2.5-tetramer thymocytes in the former group was also reduced approximately fivefold as compared with the latter ( p < 0.01 , fig .
, we found that , although the majority of the residual host - type cd4cd8 or cd4 thymocytes and cd4 splenic cells in the mixed chimeras given mhc - mismatched wild - type donor bm cells were bdc2.5-tetramer , which was similar to the control bdc2.5 mice as judged by the percentage of bdc2.5-tetramer cells ( fig .
4b and data not shown ) , the yield of the cd4cd8 and cd4cd8 bdc2.5-tetramer thymocytes as well as the splenic cd4bdc2.5-tetramer cells in those mixed chimeras was markedly reduced as compared with the control bdc2.5-nod mice ( p < 0.01 , fig . 4d 
f ) . in contrast , induction of mixed chimerism with mhc - mismatched mhc ii or mhc - matched donor bm cells did not result in any reduction of the bdc2.5-tetramer thymocytes or splenic cd4 t - cells as compared with control bdc2.5-nod mice ( fig .
these results indicate that induction of mixed chimerism with mhc - mismatched wild - type donor bm cells results in deletion of the de novo developed host - type autoreactive bdc2.5 transgenic t at cd4cd8 stage , and the deletion requires donor cell expression of mismatched mhc ii molecules .
it was reported that induction of chimerism with bm cells from mhc - mismatched nonautoimmune donors prevented t1d development ( 28,3034 ) .
however , it was not clear whether those chimeric recipients had mixed or complete chimerism , because in those reports chimerism was measured with peripheral blood mononuclear cells , and the mixed chimerism could consist of residual host - type t - cells without de novo developed host - type t - cells . to identify the true mixed chimerism , de novo developed donor- and host - type cd4cd8 thymocytes , as well as b220igm or mac1/gr1 bm resident cells , needed to be measured . therefore ,
we used a recently reported radiation - free and graft versus host disease ( gvhd ) preventative anti - cd3/cd8-based condition regimen ( 3336 ) to induce mixed or complete chimerism by transplanting graded numbers of donor cells . accordingly ,
6-week - old nod mice ( h-2k , i - a , cd45.1 ) were injected with anti - cd3 and anti - cd8 ( 5 g / g each ) on days 10 and 5 . on day 0 , the conditioned mice were intravenously injected with graded numbers ( 550  10 ) of bm and cd4 t - depleted spleen cells from mhc - mismatched c57bl/6 ( h-2k , i - a , cd45.2 ) donors .
the recipients were checked for chimerism with blood mononuclear cells and monitored weekly for t1d development for up to 100 days after hct .
thereafter , the pancreas , spleen , bm , and thymus of the recipients and control mice were harvested for evaluation of insulitis and confirmation of chimerism status . because we have reported that blood chimerism levels become stable in nod recipients conditioned with the radiation - free anti - cd3-based regimen 60 days after hct ( 34 ) , and this notion
was confirmed with representative recipients in the current study ( supplementary fig . 1 and supplementary table 1 )
we found that the development of mixed and complete chimerism was associated with donor cell dose .
in general , recipients given > 25  10 mhc - mismatched donor cells all developed complete chimerism , and the majority of recipients given < 20  10 donor cells developed mixed chimerism as judged by analysis of donor- and host - type t - cells , b - cells , and macrophages / granulocytes among blood mononuclear cells .
we also found that , 100 days after hct , 66% ( 8 of 12 ) of the control mice , but none of the mixed or complete chimeric recipients , developed t1d ( p < 0.01 , fig . 1a ) .
although almost all of the residual islets in control mice had severe insulitis , none of the islets in the mixed and complete chimeric recipients had insulitis ( p < 0.01 , fig .
1b ) , although a small portion of them showed very minor peri - insulitis ( fig .
1b ) and there was no significant difference between mixed and complete chimeric recipients ( p > 0.5 , fig .
2 . mixed and complete chimerism with mhc - mismatched bm transplants both prevented t1d in nod mice .
wild - type nod mice were conditioned with anti - cd3/cd8 ( 5 g each ) on days 10 and 5 . on day 0
, the conditioned mice were transplanted with graded doses of cd4 tcd splenocytes and whole bm from wild - type c57bl/6 donors to induce chimerism ( < 20  10 each for the mixed , 50  10 each for the complete chimeras ) .
diabetes development was monitored weekly by both urine and blood glucose for up to 100 days .
thereafter , the recipient pancreas , spleen , bm , and thymus were harvested for evaluation of insulitis and chimerism status .
c : one representative spleen cell facs profile of eight recipients examined with anti - h-2k in each group . for dc patterns ,
d : one representative facs profile of four recipients in each group examined with anti - cd45.2 and anti - cd45.1 for donor- or host - type b220igm or mac1/gr1 cells in the bm .
e : representative facs profile of four recipients in each group examined with anti - h-2k and anti - h-2k for donor- and host - type thymocytes .
furthermore , we confirmed the status of chimerism by examining the spleen , bm , and thymus of the recipients .
we observed that , in the mixed chimeras , more than one - third of the t - cells , b - cells , macrophages , and granulocytes in the spleen were host - type .
in contrast , in the complete chimeras , less than 1% of the t- and b - cells were host - type , although 20% of the macrophage and granulocytes were host - type ( fig .
we should point out that those h-2 cells may not be true host - type cells , because we found that 10% of the mac-1/gr-1 cells in the spleen of c57bl/6 mice are h-2 ( supplementary fig . 3 and supplementary table 2 ) .
the mixed chimeric recipients had both donor- and host - type b220igm immature b - cells and mac1gr1 myeloid cells in the bm , whereas the complete chimeras had only donor - type cells ( fig .
consistently , the mixed chimeric recipients had both donor- and host - type cd4cd8 thymocytes , and the complete chimeric recipients had only donor - type but no host - type thymocytes ( fig .
furthermore , after enrichment of cd11c cells from thymus , we observed donor cd11c dendritic cells ( dcs ) in the thymus of both mixed and complete chimeric recipients ( fig .
this result is consistent with a previous report showing that donor - type dcs engraft in the thymus in the chimeric recipients as judged by immunohistochemistry ( ihc ) staining ( 37 ) .
percent donor- and host - type cells in spleen of mixed and complete chimeras given mhc - mismatched bm transplants data are means  se ; n = 4 .
donor - type cells were defined as h-2k , and host - type cells were defined as h-2k . taken together , these results indicate that induction of mixed chimerism with mhc - mismatched bm transplants is as efficient as induction of complete chimerism in prevention of insulitis and t1d .
( 29 ) that induction of mixed chimerism in lethally irradiated nod recipients with bm cells from mhc - matched h-2 donors prevents t1d , but it was not clear whether those chimeric recipients were true mixed chimeras with de novo developed host - type t - cells . to clarify this issue
, we performed a similar experiment by transplanting h-2 c57bl/6 ( h-2k , i - a , cd45.2 ) donor t - cell depleted ( tcd ) bm cells ( 10  10 ) into lethally irradiated nod ( h-2k , i - a , cd45.1 ) recipients .
in addition , we induced chimerism in anti - cd3/cd8-conditioned nod recipients by transplanting bm and cd4 t - depleted spleen cells ( 50  10 each ) from h-2 c57bl/6 donors . the control nod mice were provided anti - cd3/cd8 conditioning only .
the recipients and control mice were monitored for t1d development and checked for chimerism as described above .
( 29 ) , the total body irradiation ( tbi)-conditioned nod recipients given tcd bm cells from mhc - matched h-2g7 c57bl/6 donors appeared to have so called mixed chimerism , as judged by the presence of both donor- and host - type t- and b - cells among blood mononuclear cells ( data not shown ) , and no recipients developed t1d by 100 days after hct , although 61% ( 11/18 ) of control nod given conditioning only developed t1d ( p < 0.01 , fig . 2a ) .
the anti - cd3/cd8-conditioned nod recipients from bm and cd4 t - depleted spleen cells also showed mixed chimerism among blood mononuclear cells ( data not shown ) , but 17% ( 2/12 ) of them developed t1d by 100 days after hct , although there was still a significant difference compared with nod mice given conditioning only ( p < 0.05 , fig .
2a ) . to further analyze the difference between the tbi - conditioned and the anti - cd3/cd8-conditioned chimeras , the pancreata of the chimeric recipients and control mice were evaluated for insulitis and their spleen , bm , and thymus were measured for chimerism .
we found that , although the tbi - conditioned chimeric animals had little insulitis , the anti - cd3/cd8-conditioned chimeras had severe insulitis ( p < 0.01 , fig . 2b ) and marked reduction of -cell surface as compared with the tbi - conditioned recipients ( p < 0.01 , fig .
these results indicate that insulitis is eliminated in tbi - conditioned but not in anti - cd3-conditioned chimeras .
wild - type nod mice were conditioned with anti - cd3/cd8 on days 10 and 5 . on day 0 , the conditioned mice were transplanted with cd4-tcd splenocytes and bm ( 50  10 each ) from mhc - matched h2- c57bl/6 mice to induce mixed chimerism . to induce mixed chimerism with tbi conditioning , wild - type nod
diabetes development was monitored weekly by both urine and blood glucose for up to 100 days after hct .
thereafter , the recipient pancreas , spleen , bm , and thymus were harvested for evaluation of insulitis and chimerism status .
a : diabetes development curve after hct ( n = 12 for anti - cd3-conditioned chimeric recipients ; n = 18 for conditioning alone : n = 6 for tbi - conditioned chimeric recipients ) .
b : percent insulitis ( n = 4 for anti - cd3-conditioned chimeric recipients ; n = 6 for tbi - conditioned chimeric recipients and conditioning alone ) .
c : percent -cell surface area as compared with total pancreatic tissue surface area ( n = 46 ) .
d : representative facs profiles showing spleen chimerism pattern 100 days after hct ( n = 12 ) .
for dc patterns , spleens were digested with collagenase d and enriched using cd11c microbeads .
e : representative facs profiles showing donor- or host - type b220igm or mac1/gr1 cells in the bm ( n = 4 ) .
furthermore , we found that , although there were both donor- and host - type t - cells , b - cells , and macrophage / granulocytes among the spleen cells of tbi - conditioned or anti - cd3/cd8-conditioned chimeras , the donor - type cells were dominant in the former , which was consistent with the report of beilhack et al .
( 29 ) , but the host - type cells were dominant in the latter ( fig .
in addition the immature b220igm b - cells and mac-1/gr-1 myeloid cells in the bm were almost all donor - type in the tbi - conditioned recipients , although there were both donor- and host - type cells in the anti - cd3/cd8-conditioned chimeras ( fig .
consistently , although there were both donor- and host - type thymocytes in the tbi - conditioned chimeras , there were only donor - type but no host - type cd4cd8 thymocytes in the tbi - conditioned chimeras .
in contrast , both donor- and host - type cd4cd8 thymocytes were present among the thymocytes of anti - cd3/cd8-conditioned chimeras ( fig .
finally , there was only donor - type cd11c dcs in the thymus of tbi - conditioned chimeras , although there were both donor- and host - type cd11c dcs in the anti - cd3/cd8 conditioned chimeras ( fig .
percent donor- and host - type cells in spleen of chimeras given mhc - matched bm transplants data are means  se ; n = 4 .
donor - type cells were defined as cd45.2 , and host - type cells were defined as cd45.2 . taken together , the so - called mixed chimerism in lethally irradiated tbi - conditioned chimeras consists of radiation - resistant residual host - type but no de novo developed host - type hematological cells , and they are actually complete chimeras .
in contrast , the mixed chimerism in anti - cd3/cd8-conditioned recipients consists of both residual and de novo developed host - type hematological cells , and they are truly mixed chimeras .
these results indicate that induction of mixed chimerism with mhc - matched donor bm transplants can not prevent insulitis , although induction of complete chimerism can .
we next tested whether mhc - mismatched but not mhc - matched bm transplants were able to restore thymic negative selection of autoreactive t - cells in the mixed chimeric nod mice , using t - cell receptor ( tcr ) transgenic bdc2.5-nod mice ( 38 ) .
in addition , we tested whether donor cell expression of mismatched mhc ii molecules was also required . accordingly , anti - cd3-conditioned bdc2.5-nod ( h-2k , i - a , cd45.1 ) mice were induced to develop mixed chimerism by injecting whole bm cells ( 50  10 ) from mhc - mismatched wild - type or mhc ii c57bl/6 ( h-2k , i - a , cd45.2 ) or mhc - matched h-2 c57bl/6 ( h-2k , i - a , cd45.2 ) donors . the chimerism was evaluated by fluorescence - activated cell sorting ( facs ) analysis of blood mononuclear cells as described above ( data not shown ) , and the recipients were monitored for t1d development .
we found that although 75% ( 9/12 ) of control bdc2.5-nod mice given anti - cd3 conditioning only developed t1d , none ( 0/12 ) of the mixed chimeric recipients with mhc - mismatched bm showed t1d 160 days after hct ( p < 0.01 , fig . 3a ) .
in addition , although the control mice had severe insulitis , the mixed chimeras with mhc - mismatched wild - type bm showed no insulitis ( p < 0.01 ) , although they showed mild peri - insulitis ( fig .
it is of interest that induction of mixed chimerism with mhc - mismatched mhc ii or mhc - matched h-2 donor bm cells not only failed to prevent but markedly augmented t1d development , as compared with the control bdc2.5-nod mice ( p < 0.01 , fig .
the mixed chimerism status in the different group was confirmed by flow cytometry analysis of the recipient spleen cells ( fig .
these results indicate that induction of mixed chimerism with mhc - mismatched , but not mhc - matched , donor bm cells can prevent t1d development in transgenic autoimmune bdc2.5-nod mice , and the expression of mhc ii molecules by the mhc - mismatched donor cells is required for disease prevention .
mixed chimerism with mhc - mismatched wild - type bm prevented t1d in transgenic bdc2.5-nod mice .
transgenic bdc2.5-nod mice were conditioned anti - cd3 ( 5 g ) on day 7 . on day 0 , the conditioned mice were transplanted with bm cells ( 50  10 ) from mhc - mismatched wild - type , mhc - mismatched mhc ii or mhc - matched h-2 c57bl/6 donors to induce mixed chimerism .
diabetes development was monitored weekly by both urine and blood glucose . a : diabetes development curve after hct ( n = 12 for mice given conditioning alone or recipients given bm cells from mhc - mismatched wild - type donors ; n = 7 for recipients given mhc - mismatched mhc ii donor bm cells ; n = 8 for recipients given mhc - matched bm cells ) .
b : percent insulitis for recipients given mhc mismatched wild - type bm versus conditioning alone 120 days after transplantation ( n = 4 ) .
we next tested whether there was a deletion of de novo developed host - type autoreactive t - cells in the thymus of the mixed chimeras given mhc - mismatched wild - type bm cells .
we first compared the percentage of donor- and host - type thymocytes at cd4cd8 , cd4cd8 , and cd4cd8 stages ( fig .
bdc2.5-nod mice are a cd4 t transgenic mouse line ( 38 ) , thus , we did not evaluate cd4cd8 thymocytes .
we found that host - type thymocytes were present among cd4cd8 , cd4cd8 , and cd4cd8 in anti - cd3-conditioned control bdc2.5-nod mice without hct as well as in the mixed chimeras given mhc - mismatched mhc ii or mhc matched h-2 donor bm cells .
in contrast , the host - type thymocytes in the mixed chimeras given mhc - mismatched donor bm cells were mainly present among cd4cd8 and cd4cd8 thymocytes , and they were nearly undetectable among total cd4cd8 thymocytes ( fig .
after cd45.1 host - type cells were gated on , a small percentage of cd4cd8 cells were detected in those mixed chimeric recipients , although it was 15-fold lower , as compared with control bdc2.5 mice or the mixed chimeras given mhc - mismatched mhc ii or mhc - matched donor bm cells ( p < 0.01 , fig .
interestingly , the reduction of the percentage of cd4cd8 subset among host - type thymocytes in the mixed chimeras given mhc - mismatched wild - type donor bm cells was associated a more than 20-fold increase of cd4cd8 subsets ( p < 0.01 , fig .
these results indicate that host - type cd4cd8 thymocytes in the mixed chimeras given mhc - mismatched donor bm cells are prevented to develop into cd4cd8 stage .
mixed chimerism with mhc - mismatched wild - type bm transplants mediated thymic deletion of de novo developed autoreactive t - cells .
transgenic bdc2.5-nod mice were conditioned anti - cd3 ( 5 g ) on day 7 . on day 0 ,
the conditioned mice were transplanted with bm cells ( 50  10 ) to induce mixed chimerism .
mice were analyzed for clonal deletion of autoreactive t - cells at time of diabetes development , 3060 days after hct .
a : representative facs profiles showing clonal deletion of autoreactive t - cells in the thymus .
cd45.1 host - type thymocytes were gated on and shown in cd4 versus cd8 , and cd4cd8 cells were then gated and shown in histogram of bdc2.5-tetramer ( solid line ) versus control - tetramer ( shaded area ) .
host - type cd45.1cd4 splenocytes were gated on and shown in histogram of bdc2.5-tetramer ( solid line ) versus control - tetramer ( shaded area ) ( n = 4 ) .
c  f : yield of host - type tetramer cells among cd4cd8 , cd4cd8 , and cd4cd8 thymocytes as well as cd4 splenocytes ( n = 4 ) . deletion of transgenic autoreactive thymocytes has been reported to take place at cd4cd8 stage ( 39,40 ) .
thus we tested whether this is the case in the mixed chimeric bdc2.5-nod recipients , using i - a - mimotope - tetramer ( bdc2.5-tetramer ) to identify the autoreactive bdc2.5 t - cells , as previously described ( 41 ) .
we found that 50% of the host - type cd4cd8 thymocytes in the control bdc2.5 mice given anti - cd3-conditioning only and the mixed chimeras given mismatched mhc ii or mhc - matched donor bm cells were bdc2.5-tetramer and that there was no significant difference among the three groups ( fig .
in contrast , the bdc2.5-tetramer cells among the host - type cd4cd8 thymocytes in the mixed chimeras given mhc - mismatched wild - type donor bm cells were reduced by approximately fivefold as compared with control bdc2.5-nod mice ( p < 0.01 , fig .
the yield of cd4cd8 bdc2.5-tetramer thymocytes in the former group was also reduced approximately fivefold as compared with the latter ( p < 0.01 , fig .
in addition , we found that , although the majority of the residual host - type cd4cd8 or cd4 thymocytes and cd4 splenic cells in the mixed chimeras given mhc - mismatched wild - type donor bm cells were bdc2.5-tetramer , which was similar to the control bdc2.5 mice as judged by the percentage of bdc2.5-tetramer cells ( fig .
4b and data not shown ) , the yield of the cd4cd8 and cd4cd8 bdc2.5-tetramer thymocytes as well as the splenic cd4bdc2.5-tetramer cells in those mixed chimeras was markedly reduced as compared with the control bdc2.5-nod mice ( p < 0.01 , fig . 4d 
f ) . in contrast , induction of mixed chimerism with mhc - mismatched mhc ii or mhc - matched donor bm cells did not result in any reduction of the bdc2.5-tetramer thymocytes or splenic cd4 t - cells as compared with control bdc2.5-nod mice ( fig .
these results indicate that induction of mixed chimerism with mhc - mismatched wild - type donor bm cells results in deletion of the de novo developed host - type autoreactive bdc2.5 transgenic t at cd4cd8 stage , and the deletion requires donor cell expression of mismatched mhc ii molecules .
first was our noting that induction of mixed chimerism with mhc - mismatched bm transplants from nonautoimmune donors is as effective as induction of complete chimerism in reversal of autoimmunity , elimination of insulitis , and prevention of t1d .
second , induction of mixed chimerism with mhc - matched bm transplants failed to reverse autoimmunity , eliminate insulitis , or prevent t1d development , although induction of complete chimerism with the matched bm transplants did . finally , induction of mixed chimerism with mhc - mismatched but not mhc - matched bm transplant results in thymic deletion of de novo developed host - type autoreactive t - cells , and the deletion requires donor cell expression of mhc ii molecules . by measuring the chimerism of the de novo developed thymocytes , we clearly demonstrated that induction of mixed and complete chimerism with mhc - mismatched bm transplants was equally effective in reversal of autoimmunity in nod mice .
previous reports stated that induction of so - called mixed chimerism in lethal or sublethally irradiated nod recipients with bm cells from nonautoimmune donors prevented t1d ( 28,3032 ) . in those reports , so called mixed chimerism was defined by coexistence of donor- and host - type lymphocytes in the periphery of the chimeras , but it was not clear whether the host - type lymphocytes were de novo developed after hct or residual host - type mature lymphocytes survived after tbi - conditioning . in the current study , we confirmed the mixed or complete chimerism by the presence or absence of de novo developed host - type cd4cd8 thymocytes as well as bm cells .
we observed that , although a low level ( i.e. , 1% ) of host - type cd4 or cd8 lymphocytes were present in the periphery and thymus of the complete chimeras , there were no detectable cd4cd8 thymocytes in the complete chimeras .
in contrast , both donor- and host - type cd4cd8 thymocytes were present in the thymus , and immature b- and myeloid cells were present in the bm of the mixed chimeras .
we found that , even in the true mixed chimeric recipients with mhc - mismatched bm , insulitis was eliminated and t1d was prevented .
therefore , when mhc - mismatched donor bm transplantation is used , induction of mixed chimerism is sufficient for the cure of t1d autoimmunity .
we believe this is of clinical significance since it has been reported that mixed chimeras with mhc - mismatched bm transplants provide superior immune function against infection ( 42,43 ) .
our studies also demonstrated that induction of mixed chimerism with mhc - matched bm transplants from nonautoimmune donors did not reverse autoimmunity or eliminate insulitis , although induction of complete chimerism did .
( 29 ) , we observed that induction of chimerism in lethally irradiated nod mice with tcd bm from mhc - matched h-2 c57bl/6 donors resulted in so - called mixed chimerism .
however , we found that those chimeras did not have host - type cd4cd8 thymocytes or host - type bm resident b220igm or mac1/gr1 cells , indicating that the host - type t- or b - lymphocytes were not de novo developed . therefore , those so - called mixed chimeras were in fact complete chimeras .
in contrast , we found that the true mixed chimeras with mhc - matched bm transplants had both donor- and host - type cd4cd8 thymocytes as well as b220igm and mac1/gr1 bm cells , and all of them had severe insulitis and reduced -cell quantity , indicating that induction of mixed chimerism with mhc - matched donor bm cells is not able to reverse t1d autoimmunity . using approaches similar to those that have been used to evaluate thymic deletion of autoreactive t - cells mediated by hematopoietic cell - derived antigen - presenting cells ( apcs ) in previous publications ( 18,44,45 )
, we demonstrated that induction of mixed chimerism with mhc - mismatched but not mhc - matched donor bm cells resulted in thymic deletion of de novo developed host - type autoreactive t - cells and that the deletion requires donor cell expression of mhc ii molecules .
we observed that de novo developed host - type autoreactive thymocytes in the mixed chimeric bdc2.5-nod transgenic mice given mhc mismatch but not matched c57bl/6 bm cells were deleted before the cd4cd8 thymocyte stage when they started to express tcr , and the deletion required donor cell expression of mismatched mhc ii molecules .
this indicates that autoreactive t - cell interaction with donor - type apc via tcr - mismatched - mhc ii complex play an important role in thymic negative selection of autoreactive t - cells in the mixed chimeras .
it was reported that the protection against t1d in ( bdc2.5-nod x c57bl/6 ) f1 mice ( h-2 ) was not associated with thymic deletion of the transgenic autoreactive t - cells but rather associated with positive selection of nonautoreactive t - cells expressing transgenic tcr coupling with endogenous vs ( 19 ) . however , in the mixed chimeric bdc2.5 nod mice , we found a marked reduction of nod host - type thymocytes that express autoreactive tcr ( fig .
4 ) , but no expansion of transgenic v4 coupling with endogenous vs ( supplementary fig .
positive selection of nonautoreactive host - type t - cells by mismatched mhc ii is unlikely to play a major role in reversal of autoimmunity in the mixed chimeric recipients .
it was reported that mhc ii - positive hematopoietic cells can not mediate positive selection of cd4 t - lymphocytes ( 46 ) .
it is not surprising that tolerization mechanisms in mixed chimeras are different from f1 mice . in the chimeras , the mhc ii ( i - a ) molecules are expressed by donor - type apc but not host - type thymic epithelial cells .
the strong signals from tcr interaction with allo - mhc ii may induce host - type thymocytes to go through apoptosis and result in deletion .
in contrast , in f1 mice , the mhc ii ( i - a ) molecules are expressed by both thymic epithelial cells and apcs , in which they may be able to positively select nonautoreactive thymocytes with endogenous v and unable to mediate negative selection of autoreactive transgenic thymocytes . we should also emphasize the association between failure in thymic deletion of the de novo developed host - type autoreactive t - cells and the failure in elimination of insulitis or prevention of t1d in the mixed chimeric recipients given mhc - matched h-2 donor bm transplants .
this indicates that re - establishing central tolerance in autoimmune t1d recipients is critical for prevention or reversal of autoimmunity .
however , the significant delay of t1d onset in the mixed chimeric recipients given mhc - matched h-2 donor bm transplants also indicates that peripheral tolerance mechanisms provided by nonautoimmune h-2 donor cells play a significant role in regulating t1d development , although it is not sufficient to fully prevent autoimmunity .
it was reported that non  mhc - associated loci from diabetes resistant mice mediated peripheral tolerance in autoimmune nod mice ( 16,47 ) . in summary
, we have identified the mechanisms wherein induction of mixed chimerism re - establishes the critical central tolerance in nod mice .
however , caution needs to be considered with regards to whether similar mechanisms operate in humans because of the far greater heterogeneity of this disease in humans ( 48 ) .
we recently reported that induction of mixed chimerism with mhc mismatched donor bm transplants under the radiation - free and gvhd preventative anti - cd3-based conditioning regimen can reverse new - onset t1d by augmenting residual -cell expansion ( 33 ) as well as provide immune tolerance to donor islets ( 49 ) .
in addition , allogeneic donor t - cells in transplants have been shown not to cause gvhd in humans with mixed chimerism ( 50 ) .
therefore , induction of mixed chimerism under the radiation - free anti - cd3-based conditioning regimen may represent a curative therapy for severe t1d .
 objectiveinduction of mixed or complete chimerism via hematopoietic cell transplantation ( hct ) from nonautoimmune donors could prevent or reverse type 1 diabetes ( t1d ) . in clinical settings , 
 hla - matched hct is preferred to facilitate engraftment and reduce the risk for graft versus host disease ( gvhd ) . 
 yet autoimmune t1d susceptibility is associated with certain hla types . 
 therefore , we tested whether induction of mixed chimerism with major histocompatibility complex ( mhc)-matched donors could reverse autoimmunity in the nod mouse model of t1d.research design and methodsprediabetic wild - type or transgenic bdc2.5 nod mice were conditioned with a radiation - free gvhd preventative anti - cd3/cd8 conditioning regimen and transplanted with bone marrow ( bm ) from mhc - matched or mismatched donors to induce mixed or complete chimerism . t1d 
 development and thymic deletion of host - type autoreactive t - cells in the chimeric recipients were evaluated.resultsinduction of mixed chimerism with mhc - matched nonautoimmune donor bm transplants did not prevent t1d in wild - type nod mice , although induction of complete chimerism did prevent the disease . however , induction of either mixed or complete chimerism with mhc - mismatched bm transplants prevented t1d in such mice . 
 furthermore , induction of mixed chimerism in transgenic bdc2.5-nod mice with mhc - matched or -mismatched mhc ii/ bm transplants failed to induce thymic deletion of de novo developed host - type autoreactive t - cells , whereas induction of mixed chimerism with mismatched bm transplants did.conclusionsinduction of mixed chimerism with mhc - mismatched , but not matched , donor bm transplants re - establishes thymic deletion of host - type autoreactive t - cells and prevents t1d , with donor antigen - presenting cell expression of mismatched mhc ii molecules being required . 
RESEARCH DESIGN AND METHODS
Conditioning regimen and HCT.
Flow cytometry and cell depletion/enrichment.
Pancreatic analysis.
Statistical analysis.
RESULTS
Induction of mixed and complete chimerism with BM transplants from MHC-mismatched donors both prevented insulitis and T1D development.
Induction of mixed chimerism with BM transplants from MHC-matched donors did not prevent insulitis, although induction of complete chimerism did.
Induction of mixed chimerism with BM transplants from MHC-mismatched but not matched donors resulted in thymic deletion of de novo developed host-type autoreactive T-cells.
DISCUSSION