BACKGROUND
in germinal centers , b cells undergo clonal expansion, somatic hyper-mutation in the variable region of antibody genes  <cit>  and class switch recombination  from igm to igg, iga, and ige  <cit> , processes that are dependent on helper t cells  <cit> .
antibodies to the cd <dig> epitope  have been used to identify a t cell type in germinal centers in human tonsils, spleen and lymph nodes.
these cells are cd4+ t cells  <cit> , exhibit a memory phenotype   <cit>  and are not cytolytic  <cit> .
cd57+ gc-th cells proliferate only when they are tcr-activated in the presence of il- <dig>  <cit> .
cd57+ gc-th cells express the b-cell zone homing chemokine receptor cxcr <dig> but not the t cell zone homing chemokine receptor ccr <dig>  a pattern consistent with their specific localization in gc  <cit> .
based upon their non-polarized cytokine profile, localization in gc and potential helper activity, it has been proposed that cd57+ gc-th cells may constitute a novel effector t cell subset distinct from other well known effector t cell subsets such as th <dig> and th <dig> cells  <cit> .
using a gene expression profiling study, we determined that cd57+ gc-th cells are remotely related to other memory/effector t cells in global gene expression  <cit> .
the microarray study also revealed that cd57+ gc-th cells have the unique capacity to produce cxcl <dig>  a follicle chemokine implicated in recruitment of cxcr5+ cells  <cit>  and development of follicles/gcs  <cit> .
because of their specific localization in germinal centers, the activities of cd57+ gc-th cells on b cell proliferation and antibody production have been studied by several groups of scientists  <cit> .
the results of these previous studies reveled unique features of cd57+ gc-th cells, but, when combined, they are inconclusive and widely vary from negative to neutral or positive in assessing the helper activities of cd57+ gc-th cells.
to clarify and gain more insight into their function in helping b cells, we systematically investigated the capacity of human tonsil cd57+ gc-th cells in inducing b cell ig synthesis in naïve vs. germinal center b  cells in comparison with other t cell subsets in human tonsils.
we show that cd57+ gc-th cells are more efficient than other germinal center or interfollicular t cells in supporting b cell production of ig.
cd57+ gc-th cells, when compared to other t cells, have better helper activity for gc-b cells than for naïve b cells.
cd57+ gc-th cells induced the expression of activation-induced cytosine deaminase  and csr in developing b cells.
cd40l, but not other major cytokines, is critical for the helper activity of cd57+ gc-th cells.
il- <dig> positively and tgf-β <dig> negatively regulate the helper activity of cd57+ gc-th cells.
RESULTS
distribution and identification of t helper cell subsets in tonsils
we examined the distribution of t helper cell subsets in human tonsils based upon the expression of cd <dig>  cd <dig> and cd <dig>  as reported previously  <cit> , most cd57+ cd4+ t cells are located in germinal centers surrounded by igd+ naïve b cells .
small numbers of cd57+ t cells were also present in the interfollicular areas  surrounding gc.
although some are found in ifa, cd69+ cd4+ t cells were also preferentially found in gc .
in contrast, the t cells in ifa were mostly negative for cd <dig> expression.
therefore, cd <dig>  cd <dig> and cd <dig> are useful markers to identify cd57+ gc-th cells and other t cell subsets differentially localized in tonsils: cd4+cd57+ cells , cd4+cd57-cd69+ cells , and cd4+cd57-cd69- cells .
cd57+ gc-th cells are highly efficient in supporting ig production by b cells
based upon the information obtained in figure  <dig>  the total tonsil cd4+ t cell population was fractionated into cd57+ gc-th cells , total cd57- t cells, cd57-cd69+ t cells and cd57-cd69- t cells .
we compared the b cell helping activity of cd57+ gc-th cells with that of other cd4+ t cell subsets.
we co-cultured each of the isolated t cell subsets with syngeneic tonsil cd19+ b cells in the presence of seb, a superantigen that conjugates mhc class ii molecules and tcr .
b cell receptors were cross-linked by ab to human ig μ chain and human ig  chain prior to culture to provide bcr activation signals mimicking the antigen signals in vivo.
cd57+ gc-th cells were most efficient in inducing b cell production of igm, igg, iga and ige among the t cell subsets examined.
cd57- cd69+ t cells, many of which are located in gc in a manner similar to cd57+ gc-th cells, were able to induce the production of antibodies but at significantly lower levels compared to cd57+ gc-th cells .
t cell stimulation, in this study by seb, was required for efficient induction of the b cell helper activity as it enhanced ig production up to ~ <dig> .
gc-b cells are the preferred target cells for the helper activity of cd57+ gc-th cells
because of their specific localization in germinal centers, the physiological target cells for cd57+ gc-th cells would be gc-b cells rather than naïve b cells.
we compared the helper activities of cd57+ gc-th cells and cd57- cd69+/- cd4+ t cell subsets for b cells.
in this study, we fractionated cd19+ b cells into two groups: igd+cd38- naïve b and cd38+ igd+/- gc b cells as shown in figure 2b.
cd57+ gc-th cells, when co-cultured with gc-b cells, were significantly more efficient than cd57-cd69+ t cells in inducing the production of all four isotypes of ig .
however, when co-cultured with naive b cells, cd57+ gc-th cells were not significantly different from cd57-cd69+ t cells in their induction capacity of ig .
again, the helper activities of total cd57- t cells and cd57-cd69- t cells for naïve and gc-b cells were very low.
the relative composition of igm, igg, iga and ige produced in response to cd57+ gc-th cells in the cultures with gc-b vs. naïve b cells was determined.
cd57+ gc-th cells drove the production of igm, igg, iga and ige in descending order .
class-switched ig isotypes such as igg and iga were more produced in gc-b cell cultures than in naïve b cell cultures.
there was no statistically significant difference between the two t cell subsets  in the composition of ig that they induced.
cd57+ gc-th cells induce aid expression and class switch recombination in b cells
aid expression in the maturating b cells in gc is necessary for csr and somatic hypermutation.
we examined whether cd57+ gc-th cells have the capacity to induce aid in b cells.
naïve b cells were co-cultured with cd57+ gc-th cells, and aid expression was examined .
cd57+ gc-th cells induced aid in activated b cells with peaks around days 3– <dig>  cd57+ gc-th cells were able to induce the expression of productive vhdjh-ch ig transcripts.
the major subtypes of ig transcripts in response to cd57+ gc-th cells were igg <dig>  igg <dig>  igg <dig> and iga <dig> .
when the peak expression levels of aid and the productive vhdjh-ch igg <dig> transcript  were compared, aid expression preceded the expression of igg <dig> transcript by 1– <dig> days in culture .
ig class switch recombination between tandemly repeated s regions located 5' to each ch gene generates switch circles.
we used a nested pcr technique designed to specifically detect switch circles but not genomic ig sequences.
freshly isolated gc-b, but not naïve b cells, contained switch circles, which were detected as smeared multiple bands on agarose gels as expected.
naïve b cells cultured with cd57+ gc-th cells generated detectable switch circles in a time-dependent manner .
we also used a dc-pcr technique  <cit>  to detect γ <dig> and α1/ <dig> switch circles .
again, gc-th cells induced switch circles in the naïve b cells cultured with gc-th cells.
cd40l signal is necessary for, while cytokines modulate, the helper activity of cd57+ gc-th cells
cytokines and cd40l regulate b cell maturation and ig production.
we examined whether cd40l, il- <dig>  il- <dig> and ifn-γ play any roles in the cd57+ gc-th cell-driven b cell production of ig.
in cultures with naïve b cells, the blockage of cd40l by neutralizing antibody completely suppressed the helper activity of cd57+ gc-th cells in inducing the b cell production of igm, igg <dig>  iga and ige .
in this case, igg <dig> was measured instead of total igg to avoid cross-reaction of the polyclonal capturing antibody for igg with the neutralizing antibodies to cytokines.
blockage of il- <dig> partially but specifically suppressed the production of ige, but it did not significantly suppressed other isotypes.
in contrast, blockage of ifn-γ enhanced the production of igm, igg <dig> and iga but not ige.
since cd40l is essential for the helper activity of cd57+ gc-th cells, we examined cd57+ gc-th cells and other t cells for the expression of surface cd40l.
freshly isolated cd57+ gc-th cells expressed cd40l, which became up-regulated upon t cell activation within hours , whereas cd4+cd57-cd69- interfollicular t cells did not express cd40l at significant levels.
there was no significant difference in the expression of surface cd40l between cd57+ and cd57- cd69+ cells.
in the cultures with gc-b cells, blocking of cd40l again completely suppressed the b cell helping activity of cd57+ gc-th cells .
however, il- <dig> neutralization did not significantly affect the ige production induced by cd57+ gc-th cells, an activity different from that for naïve b cells.
for gc-b cells, ifn-γ neutralization significantly increased the production of iga as it did for naive b cells.
the effects of ifn-γ neutralization on other ig isotypes were smaller.
while a slight decrease of ige production in the cultures of gc-b cells and cd57+ gc-th cells was observed, neutralization of endogenous il- <dig> did not have any statistically significant effect on cd57+ gc-th cell-driven ig production in the cultures of either naïve or gc-b cells.
exogenously-added il- <dig> enhances while tgf-β <dig> completely suppresses the b cell helping activity of cd57+ gc-th cells
to further examine the regulatory effect of cytokines, il- <dig>  il- <dig>  ifn-γ and tgf-β <dig> were exogenously added to the cultures of cd57+ gc-th cells with b cells .
in cultures of cd57+ gc-th cells with naïve b cells, exogenously added il- <dig> enhanced the production of some subsets of ig, but this effect was small and not statistically significant .
however exogenously added ifn-γ significantly suppressed the production of igg, iga and ige.
il- <dig>  when added exogenously, was highly efficient in enhancing the production of the four subsets of ig.
tgf-β <dig> completely suppressed the b cell-helping capacity of cd57+ gc-th cells for naive b cells.
in cultures of cd57+ gc-th cells with gc-b cells, il- <dig> was again highly effective in enhancing the helper activity of cd57+ gc-th cells, while tgf-β <dig> completely suppressed it .
ifn-γ partially but significantly suppressed the production of igm, igg, iga and ige.
exogenous il- <dig> added to the cultures had no effect on the cd57+ gc-th cell-driven ig production in this condition , which is in line with the negligible effect of anti-il- <dig> on gc-b cells in figure 5b.
discussion
cd57+ gc-th cells are unique cd4+ t cells.
they express the follicle homing receptor cxcr <dig> but lack the t cell area localization receptor ccr <dig>  <cit> , and reside specifically in germinal centers  <cit> .
cd57+ gc-th cells proliferate only when appropriate signals such as tcr, cd <dig> and il- <dig> are provided  <cit> .
gc-th cells are widely disseminated and diverse in their tcr sequence  <cit> .
cd57+ gc-th cells can express cd40l, icos and cxcl <dig> but are non-polarized t cells in their cytokine profile  <cit> .
it has been controversial and unclear whether cd57+ gc-th cells are intrinsically more efficient in helping b cells than other t cells or they are simply localized in germinal centers without any significant differences from other t cells in their capacity as helpers.
in this report, we systematically investigated the effector function of cd57+ gc-th cells in regulation of b cell immunoglobulin production and its regulation.
when compared for their helper activities in inducing ig synthesis by total b cells, cd57+ gc-th cells were most efficient among the t cell subsets in tonsils.
cd57+ gc-th cells were particularly more efficient in their helper activity for gc-b cells vs. naïve b cells.
cd57-cd69+ t cells were equally efficient to cd57+ gc-th cells in inducing naïve b cell differentiation for ig production, while they were less effective than cd57+ gc-th cells in helping gc-b cells.
this preference of cd57+ gc-th cells for gc-b cells is physiologically relevant, since both the helper t cell subset and target b cells are specifically present in germinal centers.
therefore, cd57+ gc-th cells would constitute an ideal t helper subset that can drive gc-b cell differentiation in germinal centers.
the effects of cytokines such as il- <dig>  il- <dig>  ifn-γ and cd40l on b cells in humans and mice have been well documented.
it is considered that cd40l is a critical factor  <cit> , and il- <dig> and il- <dig> are positive factors in regulation of b cell ig production  <cit> .
ifn-γ induces class switch to certain isotypes while it inhibits to others  <cit> .
in this study of the helper activity of cd57+ gc-th cells, the positive role of il- <dig> in promoting ig production was valid only for ige, but not igg and iga in the cultures of naïve b cells with cd57+ gc-th cells .
gc-b cells were even more resistant to the neutralization of il- <dig> than naïve b cells in cd57+ gc-th-cell driven ig production.
this smaller than expected effect of il- <dig> may be due to the fact that there is not much il- <dig> to neutralize in the cultures of gc-th cells.
this also suggests that gc-th cells may provide helper signals to gc-b cells that are not significantly affected by il- <dig>
aid  <cit>  is a molecule essential for somatic hypermutation, csr and ig gene conversion  <cit> .
we showed in this study that cd57+ gc-th cells can induce aid expression .
this capacity is consistent with their ability to induce class switch recombination, which can be detected within a few days in the cultures of naïve b cells with cd57+ gc-th cells.
cd57+ gc-th cells can induce the expression of productive igg1- <dig> and iga <dig> transcripts.
however, cd57+ gc-th cells were inefficient in induction of ige , which is consistent with their poor production capacity of il- <dig>  <cit> .
cd40l appears to be essential for the helper activity of cd57+ gc-th cells.
cd40l was required for the synthesis of all ig isotypes in all the conditions tested regardless of whether the target b cells for cd57+ gc-th cells were naïve or gc-b cells.
while neutralization of il- <dig> did not have any significant effect on the cd57+ gc-th cell-driven ig synthesis, exogenous il- <dig> was highly effective in enhancing the ig synthesis in our study.
this could be due to insufficient neutralization of the il- <dig> produced by cd57+ gc-th cells, which are known to produce il- <dig> upon tcr activation  <cit> .
another possibility is that higher concentration of il- <dig> than the level produced by cd57+ gc-th cells may be necessary to significantly enhance the ig response.
exogenous ifn-γ negatively regulates the cd57+ gc-th cell-driven ig synthesis, suggesting the potential roles of th <dig> cells or other ifn-γ producing cells in regulation of the cd57+ gc-th cells' helper activity.
tgf-β <dig> plays dual roles: it is a switch factor for iga and a potent immunosuppressive cytokine that inhibits ig synthesis  <cit> .
we did not detect any switching effect but were able to detect its suppressive activity for the cd57+ gc-th cell response.
this could be due to the fact that the culture conditions  employed in our study appear to favor the detection of the suppressive function of tgf-β.
taken together, these results imply that th <dig>  th <dig> and regulatory t cells, if present in germinal centers, could positively or negatively control the function of cd57+ gc-th cells in regulation of humoral immune responses.
indeed, there are regulatory t cells in gcs that express surface tgf-β and can effectively suppress the function of cd57+ gc-th cells  <cit> .
CONCLUSIONS
our results demonstrated the capacity of cd57+ gc-th cells in supporting csr and ig synthesis in b cells, and revealed the factors that regulate their activity, thereby substantiating the so-far inconclusive function of cd57+ gc-th cells.
the fact that these t cells have preferential and efficient helper activity for gc-b cells and are specifically localized in gcs in large numbers suggests that cd57+ gc-th cells are probably the major t helper subset responsible for supporting b cell differentiation for ig production in germinal centers.

 in germinal centers, b cells undergo clonal expansion, somatic hyper-mutation in the variable region of antibody genes and class switch recombination from igm to igg, iga, and ige, processes that are dependent on helper t cells. antibodies to the cd epitope have been used to identify a t cell type in germinal centers in human tonsils, spleen and lymph nodes. these cells are cd4+ t cells, exhibit a memory phenotype and are not cytolytic. cd57+ gc-th cells proliferate only when they are tcr-activated in the presence of il-. cd57+ gc-th cells express the b-cell zone homing chemokine receptor cxcr but not the t cell zone homing chemokine receptor ccr a pattern consistent with their specific localization in gc. based upon their non-polarized cytokine profile, localization in gc and potential helper activity, it has been proposed that cd57+ gc-th cells may constitute a novel effector t cell subset distinct from other well known effector t cell subsets such as th and th cells. using a gene expression profiling study, we determined that cd57+ gc-th cells are remotely related to other memory/effector t cells in global gene expression. the microarray study also revealed that cd57+ gc-th cells have the unique capacity to produce cxcl a follicle chemokine implicated in recruitment of cxcr5+ cells and development of follicles/gcs. because of their specific localization in germinal centers, the activities of cd57+ gc-th cells on b cell proliferation and antibody production have been studied by several groups of scientists. the results of these previous studies reveled unique features of cd57+ gc-th cells, but, when combined, they are inconclusive and widely vary from negative to neutral or positive in assessing the helper activities of cd57+ gc-th cells. to clarify and gain more insight into their function in helping b cells, we systematically investigated the capacity of human tonsil cd57+ gc-th cells in inducing b cell ig synthesis in naïve vs. germinal center b cells in comparison with other t cell subsets in human tonsils. we show that cd57+ gc-th cells are more efficient than other germinal center or interfollicular t cells in supporting b cell production of ig. cd57+ gc-th cells, when compared to other t cells, have better helper activity for gc-b cells than for naïve b cells. cd57+ gc-th cells induced the expression of activation-induced cytosine deaminase and csr in developing b cells. cd40l, but not other major cytokines, is critical for the helper activity of cd57+ gc-th cells. il- positively and tgf-β negatively regulate the helper activity of cd57+ gc-th cells.  distribution and identification of t helper cell subsets in tonsils we examined the distribution of t helper cell subsets in human tonsils based upon the expression of cd cd and cd as reported previously, most cd57+ cd4+ t cells are located in germinal centers surrounded by igd+ naïve b cells. small numbers of cd57+ t cells were also present in the interfollicular areas surrounding gc. although some are found in ifa, cd69+ cd4+ t cells were also preferentially found in gc. in contrast, the t cells in ifa were mostly negative for cd expression. therefore, cd cd and cd are useful markers to identify cd57+ gc-th cells and other t cell subsets differentially localized in tonsils: cd4+cd57+ cells, cd4+cd57-cd69+ cells, and cd4+cd57-cd69- cells. cd57+ gc-th cells are highly efficient in supporting ig production by b cells based upon the information obtained in figure the total tonsil cd4+ t cell population was fractionated into cd57+ gc-th cells, total cd57- t cells, cd57-cd69+ t cells and cd57-cd69- t cells. we compared the b cell helping activity of cd57+ gc-th cells with that of other cd4+ t cell subsets. we co-cultured each of the isolated t cell subsets with syngeneic tonsil cd19+ b cells in the presence of seb, a superantigen that conjugates mhc class ii molecules and tcr. b cell receptors were cross-linked by ab to human ig μ chain and human ig chain prior to culture to provide bcr activation signals mimicking the antigen signals in vivo. cd57+ gc-th cells were most efficient in inducing b cell production of igm, igg, iga and ige among the t cell subsets examined. cd57- cd69+ t cells, many of which are located in gc in a manner similar to cd57+ gc-th cells, were able to induce the production of antibodies but at significantly lower levels compared to cd57+ gc-th cells. t cell stimulation, in this study by seb, was required for efficient induction of the b cell helper activity as it enhanced ig production up to ~. gc-b cells are the preferred target cells for the helper activity of cd57+ gc-th cells because of their specific localization in germinal centers, the physiological target cells for cd57+ gc-th cells would be gc-b cells rather than naïve b cells. we compared the helper activities of cd57+ gc-th cells and cd57- cd69+/- cd4+ t cell subsets for b cells. in this study, we fractionated cd19+ b cells into two groups: igd+cd38- naïve b and cd38+ igd+/- gc b cells as shown in figure 2b. cd57+ gc-th cells, when co-cultured with gc-b cells, were significantly more efficient than cd57-cd69+ t cells in inducing the production of all four isotypes of ig. however, when co-cultured with naive b cells, cd57+ gc-th cells were not significantly different from cd57-cd69+ t cells in their induction capacity of ig. again, the helper activities of total cd57- t cells and cd57-cd69- t cells for naïve and gc-b cells were very low. the relative composition of igm, igg, iga and ige produced in response to cd57+ gc-th cells in the cultures with gc-b vs. naïve b cells was determined. cd57+ gc-th cells drove the production of igm, igg, iga and ige in descending order. class-switched ig isotypes such as igg and iga were more produced in gc-b cell cultures than in naïve b cell cultures. there was no statistically significant difference between the two t cell subsets in the composition of ig that they induced. cd57+ gc-th cells induce aid expression and class switch recombination in b cells aid expression in the maturating b cells in gc is necessary for csr and somatic hypermutation. we examined whether cd57+ gc-th cells have the capacity to induce aid in b cells. naïve b cells were co-cultured with cd57+ gc-th cells, and aid expression was examined. cd57+ gc-th cells induced aid in activated b cells with peaks around days 3– cd57+ gc-th cells were able to induce the expression of productive vhdjh-ch ig transcripts. the major subtypes of ig transcripts in response to cd57+ gc-th cells were igg igg igg and iga. when the peak expression levels of aid and the productive vhdjh-ch igg transcript were compared, aid expression preceded the expression of igg transcript by 1– days in culture. ig class switch recombination between tandemly repeated s regions located 5' to each ch gene generates switch circles. we used a nested pcr technique designed to specifically detect switch circles but not genomic ig sequences. freshly isolated gc-b, but not naïve b cells, contained switch circles, which were detected as smeared multiple bands on agarose gels as expected. naïve b cells cultured with cd57+ gc-th cells generated detectable switch circles in a time-dependent manner. we also used a dc-pcr technique to detect γ and α1/ switch circles. again, gc-th cells induced switch circles in the naïve b cells cultured with gc-th cells. cd40l signal is necessary for, while cytokines modulate, the helper activity of cd57+ gc-th cells cytokines and cd40l regulate b cell maturation and ig production. we examined whether cd40l, il- il- and ifn-γ play any roles in the cd57+ gc-th cell-driven b cell production of ig. in cultures with naïve b cells, the blockage of cd40l by neutralizing antibody completely suppressed the helper activity of cd57+ gc-th cells in inducing the b cell production of igm, igg iga and ige. in this case, igg was measured instead of total igg to avoid cross-reaction of the polyclonal capturing antibody for igg with the neutralizing antibodies to cytokines. blockage of il- partially but specifically suppressed the production of ige, but it did not significantly suppressed other isotypes. in contrast, blockage of ifn-γ enhanced the production of igm, igg and iga but not ige. since cd40l is essential for the helper activity of cd57+ gc-th cells, we examined cd57+ gc-th cells and other t cells for the expression of surface cd40l. freshly isolated cd57+ gc-th cells expressed cd40l, which became up-regulated upon t cell activation within hours, whereas cd4+cd57-cd69- interfollicular t cells did not express cd40l at significant levels. there was no significant difference in the expression of surface cd40l between cd57+ and cd57- cd69+ cells. in the cultures with gc-b cells, blocking of cd40l again completely suppressed the b cell helping activity of cd57+ gc-th cells. however, il- neutralization did not significantly affect the ige production induced by cd57+ gc-th cells, an activity different from that for naïve b cells. for gc-b cells, ifn-γ neutralization significantly increased the production of iga as it did for naive b cells. the effects of ifn-γ neutralization on other ig isotypes were smaller. while a slight decrease of ige production in the cultures of gc-b cells and cd57+ gc-th cells was observed, neutralization of endogenous il- did not have any statistically significant effect on cd57+ gc-th cell-driven ig production in the cultures of either naïve or gc-b cells. exogenously-added il- enhances while tgf-β completely suppresses the b cell helping activity of cd57+ gc-th cells to further examine the regulatory effect of cytokines, il- il- ifn-γ and tgf-β were exogenously added to the cultures of cd57+ gc-th cells with b cells. in cultures of cd57+ gc-th cells with naïve b cells, exogenously added il- enhanced the production of some subsets of ig, but this effect was small and not statistically significant. however exogenously added ifn-γ significantly suppressed the production of igg, iga and ige. il- when added exogenously, was highly efficient in enhancing the production of the four subsets of ig. tgf-β completely suppressed the b cell-helping capacity of cd57+ gc-th cells for naive b cells. in cultures of cd57+ gc-th cells with gc-b cells, il- was again highly effective in enhancing the helper activity of cd57+ gc-th cells, while tgf-β completely suppressed it. ifn-γ partially but significantly suppressed the production of igm, igg, iga and ige. exogenous il- added to the cultures had no effect on the cd57+ gc-th cell-driven ig production in this condition, which is in line with the negligible effect of anti-il- on gc-b cells in figure 5b. discussion cd57+ gc-th cells are unique cd4+ t cells. they express the follicle homing receptor cxcr but lack the t cell area localization receptor ccr, and reside specifically in germinal centers. cd57+ gc-th cells proliferate only when appropriate signals such as tcr, cd and il- are provided. gc-th cells are widely disseminated and diverse in their tcr sequence. cd57+ gc-th cells can express cd40l, icos and cxcl but are non-polarized t cells in their cytokine profile. it has been controversial and unclear whether cd57+ gc-th cells are intrinsically more efficient in helping b cells than other t cells or they are simply localized in germinal centers without any significant differences from other t cells in their capacity as helpers. in this report, we systematically investigated the effector function of cd57+ gc-th cells in regulation of b cell immunoglobulin production and its regulation. when compared for their helper activities in inducing ig synthesis by total b cells, cd57+ gc-th cells were most efficient among the t cell subsets in tonsils. cd57+ gc-th cells were particularly more efficient in their helper activity for gc-b cells vs. naïve b cells. cd57-cd69+ t cells were equally efficient to cd57+ gc-th cells in inducing naïve b cell differentiation for ig production, while they were less effective than cd57+ gc-th cells in helping gc-b cells. this preference of cd57+ gc-th cells for gc-b cells is physiologically relevant, since both the helper t cell subset and target b cells are specifically present in germinal centers. therefore, cd57+ gc-th cells would constitute an ideal t helper subset that can drive gc-b cell differentiation in germinal centers. the effects of cytokines such as il- il- ifn-γ and cd40l on b cells in humans and mice have been well documented. it is considered that cd40l is a critical factor, and il- and il- are positive factors in regulation of b cell ig production. ifn-γ induces class switch to certain isotypes while it inhibits to others. in this study of the helper activity of cd57+ gc-th cells, the positive role of il- in promoting ig production was valid only for ige, but not igg and iga in the cultures of naïve b cells with cd57+ gc-th cells. gc-b cells were even more resistant to the neutralization of il- than naïve b cells in cd57+ gc-th-cell driven ig production. this smaller than expected effect of il- may be due to the fact that there is not much il- to neutralize in the cultures of gc-th cells. this also suggests that gc-th cells may provide helper signals to gc-b cells that are not significantly affected by il- aid is a molecule essential for somatic hypermutation, csr and ig gene conversion. we showed in this study that cd57+ gc-th cells can induce aid expression. this capacity is consistent with their ability to induce class switch recombination, which can be detected within a few days in the cultures of naïve b cells with cd57+ gc-th cells. cd57+ gc-th cells can induce the expression of productive igg1- and iga transcripts. however, cd57+ gc-th cells were inefficient in induction of ige, which is consistent with their poor production capacity of il-. cd40l appears to be essential for the helper activity of cd57+ gc-th cells. cd40l was required for the synthesis of all ig isotypes in all the conditions tested regardless of whether the target b cells for cd57+ gc-th cells were naïve or gc-b cells. while neutralization of il- did not have any significant effect on the cd57+ gc-th cell-driven ig synthesis, exogenous il- was highly effective in enhancing the ig synthesis in our study. this could be due to insufficient neutralization of the il- produced by cd57+ gc-th cells, which are known to produce il- upon tcr activation. another possibility is that higher concentration of il- than the level produced by cd57+ gc-th cells may be necessary to significantly enhance the ig response. exogenous ifn-γ negatively regulates the cd57+ gc-th cell-driven ig synthesis, suggesting the potential roles of th cells or other ifn-γ producing cells in regulation of the cd57+ gc-th cells' helper activity. tgf-β plays dual roles: it is a switch factor for iga and a potent immunosuppressive cytokine that inhibits ig synthesis. we did not detect any switching effect but were able to detect its suppressive activity for the cd57+ gc-th cell response. this could be due to the fact that the culture conditions employed in our study appear to favor the detection of the suppressive function of tgf-β. taken together, these results imply that th th and regulatory t cells, if present in germinal centers, could positively or negatively control the function of cd57+ gc-th cells in regulation of humoral immune responses. indeed, there are regulatory t cells in gcs that express surface tgf-β and can effectively suppress the function of cd57+ gc-th cells.  our results demonstrated the capacity of cd57+ gc-th cells in supporting csr and ig synthesis in b cells, and revealed the factors that regulate their activity, thereby substantiating the so-far inconclusive function of cd57+ gc-th cells. the fact that these t cells have preferential and efficient helper activity for gc-b cells and are specifically localized in gcs in large numbers suggests that cd57+ gc-th cells are probably the major t helper subset responsible for supporting b cell differentiation for ig production in germinal centers. 
the function of cd57+ cd4+ t cells, constituting a major subset of germinal center t cells in human lymphoid tissues, has been unclear. cd57+ gc-th cells were particularly more efficient than other t cells in helping gc-b cells but not naïve b cells. cd57+cd4+ t cells in the germinal centers of human lymphoid tissues are the major t helper cell subset for gc-b cells in ig synthesis. we demonstrated that cd57+ gc-th cells are highly efficient in helping b cell production of all four subsets of ig compared to other t-helper cells located in germinal centers or interfollicular areas. furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. however, il- when added exogenously, significantly enhanced the helper activity of cd57+ gc-th cells, while tgf-β completely and ifn-γ partially suppressed the cd57+ gc-th cell-driven ig production. their helper activity is consistent with their capacity to induce aid and class switch recombination, and can be regulated by cd40l, il- il- and tgf-β. to clarify and gain more insight into their function in helping b cells, we systematically investigated the capacity of human tonsil cd57+ gc-th cells in inducing b cell ig synthesis. cd57+ gc-th cells induced the expression of activation-induced cytosine deaminase and class switch recombination in developing b cells. there have been contradictory reports regarding the b cell helping function of cd57+ gc-th cells in production of immunoglobulin. cd40l, but not il- il- and ifn-γ, was critical in cd57+ gc-th cell-driven b cell production of ig. igg1- and iga were the major ig isotypes induced by cd57+ gc-th cells.
 the function of cd57+ cd4+ t cells, constituting a major subset of germinal center t cells in human lymphoid tissues, has been unclear. there have been contradictory reports regarding the b cell helping function of cd57+ gc-th cells in production of immunoglobulin. furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. to clarify and gain more insight into their function in helping b cells, we systematically investigated the capacity of human tonsil cd57+ gc-th cells in inducing b cell ig synthesis.  we demonstrated that cd57+ gc-th cells are highly efficient in helping b cell production of all four subsets of ig compared to other t-helper cells located in germinal centers or interfollicular areas. cd57+ gc-th cells were particularly more efficient than other t cells in helping gc-b cells but not naïve b cells. cd57+ gc-th cells induced the expression of activation-induced cytosine deaminase and class switch recombination in developing b cells. igg1- and iga were the major ig isotypes induced by cd57+ gc-th cells. cd40l, but not il- il- and ifn-γ, was critical in cd57+ gc-th cell-driven b cell production of ig. however, il- when added exogenously, significantly enhanced the helper activity of cd57+ gc-th cells, while tgf-β completely and ifn-γ partially suppressed the cd57+ gc-th cell-driven ig production.  cd57+cd4+ t cells in the germinal centers of human lymphoid tissues are the major t helper cell subset for gc-b cells in ig synthesis. their helper activity is consistent with their capacity to induce aid and class switch recombination, and can be regulated by cd40l, il- il- and tgf-β. 