BACKGROUND
from the people infected worldwide with mtb only about 5-10% develope disease, while the majority can control the disease through nk cell and th <dig> immune responses characterized by the release of ifn-γ and tnf leading to the cytotoxic activity of cd8+ t cells and macrophages.
susceptibility to mtb is considered to result from a suboptimal immune response and therefore a vaccine against mtb would improve this status.
however, no vaccine based on mtb is currently available.
bcg as an attenuated strain of m. bovis has been used as a vaccine against mtb but without achieving a reliable protection  <cit> .
thus, alternative vaccination strategies are urgently needed  <cit> .
currently modified bcg vaccines are the most common tested in clinical trials but also few selective mtb antigens have been tested for their capacity to stimulate immune responses in order to use them as a vaccine  <cit> .
a successful vaccine should induce strong cd <dig> and th <dig> memory responses and at the same time avoid the induction of immune tolerance mechanisms.
immune deviation towards th <dig> responses is a hallmark of many infections leading to microbial persistence  <cit> .
thus, we wanted to investigate whether immune deviation could be detected by selective mtb components.
we studied these factors not as antigens presented on mhc molecules but as factors to induce dc maturation.
the quality of dc maturation was then assessed as well as the dc-mediated immune responses of cd4+ t cells.
initially, all types of dc maturation were believed to induce dc immunogenicity.
by establishing a semi-mature stage of dc maturation we could demonstrate that matured dc nevertheless could act tolerogenic.
tnf treatment of murine bone marrow-derived dc led to their partial maturation and after i.v.
injection induced protective il- <dig> producing t cells  in the model of experimental autoimmune encephalomyelitis   <cit> .
this effect was antigen-specific and not dependent on bystander proteins  <cit> .
cytokine analysis revealed that also il- <dig> and il- <dig> produced from cd4+ t and nkt cells contributed to the protection, indicating a th <dig> cell involvement  <cit> .
interestingly, although injections of tnf/dc induced a mixed tr1/th <dig> response when injected alone, antigen-specific pre-treatment of mice with tnf/dc did not boost subsequent th <dig> cell responses such as leishamania major infection of balb/c mice  <cit>  or allergic asthma  <cit> .
this effect of tolerogenic mature dc is not restricted to the murine system.
others showed that tnf/pge2-maturation of human monocyte-derived dc was required to perform cross-tolerance  <cit> .
thus, dc maturation must not necessarily indicate the induction of protective immunity.
membrane and secreted molecules but also whole protein extracts of mtb represent promising candidates in mtb vaccine development.
the ra and rv strains have been studied extensively and recent gene array analysis indicates that the rv strain is by far better in promoting th <dig> responses  <cit> .
in this study, culture filtrate proteins  isolated from mtb h37rv and h37ra strains  <cit>  were compared to find out whether attenuation versus virulence would induce differences in cd4+ t cell polarization.
the 38-kda protein represents an immunodominant phosphate-binding protein that was originally identified in pulmonary tuberculosis and a model antigen to screen for mtb infections  <cit> .
in mycobacterial culture fluid the antigen  <dig> complex b  is a major secretory component of mtb that is also considered as a candidate for a vaccine due to its protection in animal experiments.
the already commercially available 30-kda protein is part of the ag85b complex and when expressed in bcg shows more potent protection against mtb  <cit> .
thus cfp preparations as well as these two immunodominant secretory proteins 30-kda and 38-kda antigens derived from the virulent strain mtb h37rv  <cit>  may represent candidates for vaccine development.
since effective anti-mycobacterial immune responses are of the th <dig> and not th <dig> type, we developed a human cd4+ t cell polarization system to test these antigens for their potential to shift immune responses towards th <dig> as a sign of immune evasion.
here we addressed the questions whether the 30-kda, 38-kda or cfp preparations from h37rv and h37ra were able to mount dc maturation in vitro and to instruct the dc for a subsequent polarization of cd4+ t-helper  cell responses.
to enable the polarization of a substantial frequency of cd4+ t cells, we used the superantigen seb.
the results indicate a partial activation/maturation of dc as detected by surface markers and cytokine production.
the 30-kda and 38-kda mtb antigens appeared weaker dc stimulators than the ra and rv preparations.
primary t cell stimulations by these dc revealed similar proliferation rates.
however, the secondary stimulation leading to polarized cd4+ t cell responses showed some differences.
while robust ifn-γ production could be observed by all mtb preparations, it was of note that the 30 kda and 38 kda antigens also maintained il- <dig> production as a sign of immune deviation.
RESULTS
the 30-kda and 38-kda mtb antigens induce only a mild upregulation of dc maturation markers and cytokine production
various mycobacterial components induce maturation of dcs but mostly mtb surface products have been investigated  <cit> .
the immunogenicity of the different mtb preparations was tested by their incubation with human monocyte-derived dc in vitro.
after 24 h of stimulation positive control dc that were stimulated with lps, strongly upregulated the key surface markers cd <dig>  cd <dig> and cd <dig> .
the mtb preparations h37rv and h37ra showed a low stimulatory capacity and the 30-kda and 38-kda preparations had only a weak or even no effect on these markers .
in comparison to the strong maturation by mtb surface products that was similar to lps  <cit> , only a mild upregulation of typical maturation markers was observed here.
to further test the dc maturation capacity of mtb antigens, supernatants of the stimulated cells were analysed for their cytokine content.
surprisingly, only the lps control revealed measurable amounts of il-12p <dig> but not the mycobacterial antigens .
while il- <dig> release by dcs was variable and highly donor-dependent, il- <dig> production could be observed at low amounts by lps, h37rv and h37ra .
consistently il-12p <dig> was induced by lps, h37rv and h37ra but only at low concentrations of below 1 ng/ml by the 30-kda and 38-kda antigens .
these data indicate that especially the 30-kda and 38-kda preparations induce only a partial dc maturation that may lead to a tolerogenic or immune deviatory cd4+ th <dig> cell response as observed in murine models  <cit> .
in fact, more recent gene profiling experiments indicate that murine dcs matured only partially e.g.
with tnf or trypanosoma brucei antigens show a common signature of only  <dig> regulated inflammatory genes identified for th2-inducing dcs, whereas the same gene set as well as additional other genes induced by bacterial lps result in full dc maturation and th <dig> induction  <cit> .
interstingly, after restimulation of peripheral blood cells from patients with recurrent tuberculosis with the 30-kda antigen we found depressed levels of il- <dig> previously  <cit> .
in the following experiments we addressed the question whether such quantitative differences in dc maturation may affect t cell priming capacity or the polarization into th1/th <dig> responses.
from our murine data we hypothesized that also partial human dc maturation as observed for the 30-kda and 38-kda preparations may lead to minor differences in the t cell proliferation but significantly alter th1/th <dig> cytokine profiles.
similar allogeneic and seb-dependent t cell priming capacity by the differentially mtb-matured dc
we then analysed whether the differential dc maturation conditions would result in diverging t cell priming capacities.
when the differentially matured dcs were used to prime allogeneic t cells in vitro all stimuli appeared remarkably similar to the lps control .
similar proliferation rates were also obtained by stimulating syngeneic t cells with the differentially matured dcs and the superantigen seb .
thus, despite strong differences on dc maturation markers and cytokine profiles, the capacities to stimulate primary t cells did not differ substantially between the matured dc.
the discrepancies between diverging surface marker expressions by dcs but similar t cell proliferation rates may indicate that not the quantity of the t cell response but the quality may have changed.
former observations in the murine system showed that dcs that were stimulated with trypanosoma antigens induced very similar proliferation rates of t cells after injection in vivo <cit> .
however, the qualities of t helper cell polarization differed markedly.
therefore we set up a cd4+ t cell proliferation assay where polarization of t cells could be tested.
the 30-kda and 38-kda mtb antigens maintain th <dig> cell responses
previously we found that a quantitatively weaker t cell stimulation by dc may result in the same t cell proliferation rates but different polarizations into cd4+ t helper  cell subsets  <cit> .
since the frequency of naive antigen-specific t cells from pbmcs is too low to be followed by any mhc ii-restricted peptide stimulation in vitro, we used a superantigen system where higher naive antigen-specific frequencies of t cells can be activated during t cell priming  <cit> .
in this system we explored the th <dig> or th <dig> subset polarization of the mtb antigen-matured dc.
primary seb-stimulated t cells were restimulated after 7 days with pma/ionomycin and their supernatants tested for cytokine release.
all groups of stimulated t cells released substantial amounts of ifn-γ and il- <dig> with no significant differences .
however, t cells that were stimulated by 30-kda- or 38-kda-matured dc maintained a high il- <dig> release as compared to immature  dc, although the 30-kda protein showed only as a trend .
when the ifn-γ/il- <dig> ratio was calculated the 38-kda antigen showed the strongest il- <dig> bias although no significance was achieved by this calculation .
these data indicate that dcs matured with the 38-kda antigen and as a trend also with the 30-kda antigen, allow seb-specific t cell responses biased towards a th <dig> profile, similar to immature  dcs.
although immune responses tested at the level of dcs against intracellular mtb and many of their antigens are dominated by ifn-γ producing cd8+ killer t cell and th <dig> responses  <cit> , also th <dig> responses have been observed after infection of human monocytes with bcg prior to differentiation into dcs  <cit> .
treatment of pbmcs with peptides derived from the regions of difference  antigens  <dig>   <dig> and  <dig> expressed by mtb but not bcg led to th <dig> cytokine production  <cit> .
moreover, the presence of regulatory t cells and th <dig> cells may be lined to infection susceptibility to mtb  <cit> .
in fact, patients suffering from diabetes mellitus are more susceptible to tuberculosis development and this could be linked to a th2-bias of their anti-mtb responses  <cit> .
we provide evidence for two novel mtb antigens  that induce th <dig> responses in vitro in spite of their capacity to elicit only incomplete dc maturation.
CONCLUSIONS
here we showed that the 30-kda and 38-kda antigens as mtb components induce only a partial dc maturation leading to a th <dig> shift in t cell polarization assays.
since efficient immune protection from intracellular bacteria such as mtb depends on ifn-γ dependent cd4+ th <dig> and cytotoxic cd8+ t cell responses, the observed th <dig> shift can be envisaged as an immune escape mechanism by mtb.
thus, using the two 30-kda and 38-kda antigens as an mtb vaccines may lead to a similar th <dig> shift in vivo.
therefore these findings may have implications for vaccination strategies against mtb  <cit>  or for the pathological outcomes observed in mtb infected individuals suffering also from other diseases such as diabetes  <cit> .
although mtb interferes with several innate immune receptors to raise an immune response  <cit>  it has developed several mechanisms of immune evasion strategies to prevent elimination  <cit> .
although infected individuals generate immune responses and most people can control the infection they never reach complete mtb elimination and microbial sterility  <cit> .
thus, in most cases mtb infections represent a fine balance between immune reaction and immune evasion.
the latter mechanisms may also be envisaged to prevent immune pathology by too strong immune reactions.
together this reflects the co-evolution strategy of mtb  <cit> .
to develop effective vaccines without strong side effects this fine balance and all participating mechanisms have to be considered.
a large part of the immune evasion by mtb is based on the persistent or latent infection of macrophages and the tlr-2-dependent down-regulation of antigen presenting function by mtb in these macrophages  <cit> .
the emerging role of dcs as regulators of tuberculosis disease has been elucidated only during the last years by genome-wide transcriptome analyses  <cit> .
it is well possible that also the induction of suboptimal maturation of dcs reflects another mechanism of mtb to target antigen presenting cells.
in our system we studied selective mtb antigens and found that the two antigens 30-kda and 38-kda of mtb may be involved in immune evasion at the level of dcs.
the quality of dc manipulation by mtb appears to differ from what is known from macrophages.
while for macrophages the mhc-dependent antigen presenting function seems to dominate our data point to an intact antigen presentation as observed in the allo-mlr and seb-priming, but a bias towards th <dig> immunity.
these in vitro data require now further validation in vivo but may indicate that the 30-kda and 38-kda antigens represent suboptimal mtb vaccine candidates.

 from the people infected worldwide with mtb only about 5-10% develope disease, while the majority can control the disease through nk cell and th immune responses characterized by the release of ifn-γ and tnf leading to the cytotoxic activity of cd8+ t cells and macrophages. susceptibility to mtb is considered to result from a suboptimal immune response and therefore a vaccine against mtb would improve this status. however, no vaccine based on mtb is currently available. bcg as an attenuated strain of m. bovis has been used as a vaccine against mtb but without achieving a reliable protection. thus, alternative vaccination strategies are urgently needed. currently modified bcg vaccines are the most common tested in clinical trials but also few selective mtb antigens have been tested for their capacity to stimulate immune responses in order to use them as a vaccine. a successful vaccine should induce strong cd and th memory responses and at the same time avoid the induction of immune tolerance mechanisms. immune deviation towards th responses is a hallmark of many infections leading to microbial persistence. thus, we wanted to investigate whether immune deviation could be detected by selective mtb components. we studied these factors not as antigens presented on mhc molecules but as factors to induce dc maturation. the quality of dc maturation was then assessed as well as the dc-mediated immune responses of cd4+ t cells. initially, all types of dc maturation were believed to induce dc immunogenicity. by establishing a semi-mature stage of dc maturation we could demonstrate that matured dc nevertheless could act tolerogenic. tnf treatment of murine bone marrow-derived dc led to their partial maturation and after i.v. injection induced protective il- producing t cells in the model of experimental autoimmune encephalomyelitis. this effect was antigen-specific and not dependent on bystander proteins. cytokine analysis revealed that also il- and il- produced from cd4+ t and nkt cells contributed to the protection, indicating a th cell involvement. interestingly, although injections of tnf/dc induced a mixed tr1/th response when injected alone, antigen-specific pre-treatment of mice with tnf/dc did not boost subsequent th cell responses such as leishamania major infection of balb/c mice or allergic asthma. this effect of tolerogenic mature dc is not restricted to the murine system. others showed that tnf/pge2-maturation of human monocyte-derived dc was required to perform cross-tolerance. thus, dc maturation must not necessarily indicate the induction of protective immunity. membrane and secreted molecules but also whole protein extracts of mtb represent promising candidates in mtb vaccine development. the ra and rv strains have been studied extensively and recent gene array analysis indicates that the rv strain is by far better in promoting th responses. in this study, culture filtrate proteins isolated from mtb h37rv and h37ra strains were compared to find out whether attenuation versus virulence would induce differences in cd4+ t cell polarization. the 38-kda protein represents an immunodominant phosphate-binding protein that was originally identified in pulmonary tuberculosis and a model antigen to screen for mtb infections. in mycobacterial culture fluid the antigen complex b is a major secretory component of mtb that is also considered as a candidate for a vaccine due to its protection in animal experiments. the already commercially available 30-kda protein is part of the ag85b complex and when expressed in bcg shows more potent protection against mtb. thus cfp preparations as well as these two immunodominant secretory proteins 30-kda and 38-kda antigens derived from the virulent strain mtb h37rv may represent candidates for vaccine development. since effective anti-mycobacterial immune responses are of the th and not th type, we developed a human cd4+ t cell polarization system to test these antigens for their potential to shift immune responses towards th as a sign of immune evasion. here we addressed the questions whether the 30-kda, 38-kda or cfp preparations from h37rv and h37ra were able to mount dc maturation in vitro and to instruct the dc for a subsequent polarization of cd4+ t-helper cell responses. to enable the polarization of a substantial frequency of cd4+ t cells, we used the superantigen seb. the results indicate a partial activation/maturation of dc as detected by surface markers and cytokine production. the 30-kda and 38-kda mtb antigens appeared weaker dc stimulators than the ra and rv preparations. primary t cell stimulations by these dc revealed similar proliferation rates. however, the secondary stimulation leading to polarized cd4+ t cell responses showed some differences. while robust ifn-γ production could be observed by all mtb preparations, it was of note that the 30 kda and 38 kda antigens also maintained il- production as a sign of immune deviation.  the 30-kda and 38-kda mtb antigens induce only a mild upregulation of dc maturation markers and cytokine production various mycobacterial components induce maturation of dcs but mostly mtb surface products have been investigated. the immunogenicity of the different mtb preparations was tested by their incubation with human monocyte-derived dc in vitro. after 24 h of stimulation positive control dc that were stimulated with lps, strongly upregulated the key surface markers cd cd and cd. the mtb preparations h37rv and h37ra showed a low stimulatory capacity and the 30-kda and 38-kda preparations had only a weak or even no effect on these markers. in comparison to the strong maturation by mtb surface products that was similar to lps, only a mild upregulation of typical maturation markers was observed here. to further test the dc maturation capacity of mtb antigens, supernatants of the stimulated cells were analysed for their cytokine content. surprisingly, only the lps control revealed measurable amounts of il-12p but not the mycobacterial antigens. while il- release by dcs was variable and highly donor-dependent, il- production could be observed at low amounts by lps, h37rv and h37ra. consistently il-12p was induced by lps, h37rv and h37ra but only at low concentrations of below 1 ng/ml by the 30-kda and 38-kda antigens. these data indicate that especially the 30-kda and 38-kda preparations induce only a partial dc maturation that may lead to a tolerogenic or immune deviatory cd4+ th cell response as observed in murine models. in fact, more recent gene profiling experiments indicate that murine dcs matured only partially e.g. with tnf or trypanosoma brucei antigens show a common signature of only regulated inflammatory genes identified for th2-inducing dcs, whereas the same gene set as well as additional other genes induced by bacterial lps result in full dc maturation and th induction. interstingly, after restimulation of peripheral blood cells from patients with recurrent tuberculosis with the 30-kda antigen we found depressed levels of il- previously. in the following experiments we addressed the question whether such quantitative differences in dc maturation may affect t cell priming capacity or the polarization into th1/th responses. from our murine data we hypothesized that also partial human dc maturation as observed for the 30-kda and 38-kda preparations may lead to minor differences in the t cell proliferation but significantly alter th1/th cytokine profiles. similar allogeneic and seb-dependent t cell priming capacity by the differentially mtb-matured dc we then analysed whether the differential dc maturation conditions would result in diverging t cell priming capacities. when the differentially matured dcs were used to prime allogeneic t cells in vitro all stimuli appeared remarkably similar to the lps control. similar proliferation rates were also obtained by stimulating syngeneic t cells with the differentially matured dcs and the superantigen seb. thus, despite strong differences on dc maturation markers and cytokine profiles, the capacities to stimulate primary t cells did not differ substantially between the matured dc. the discrepancies between diverging surface marker expressions by dcs but similar t cell proliferation rates may indicate that not the quantity of the t cell response but the quality may have changed. former observations in the murine system showed that dcs that were stimulated with trypanosoma antigens induced very similar proliferation rates of t cells after injection in vivo. however, the qualities of t helper cell polarization differed markedly. therefore we set up a cd4+ t cell proliferation assay where polarization of t cells could be tested. the 30-kda and 38-kda mtb antigens maintain th cell responses previously we found that a quantitatively weaker t cell stimulation by dc may result in the same t cell proliferation rates but different polarizations into cd4+ t helper cell subsets. since the frequency of naive antigen-specific t cells from pbmcs is too low to be followed by any mhc ii-restricted peptide stimulation in vitro, we used a superantigen system where higher naive antigen-specific frequencies of t cells can be activated during t cell priming. in this system we explored the th or th subset polarization of the mtb antigen-matured dc. primary seb-stimulated t cells were restimulated after 7 days with pma/ionomycin and their supernatants tested for cytokine release. all groups of stimulated t cells released substantial amounts of ifn-γ and il- with no significant differences. however, t cells that were stimulated by 30-kda- or 38-kda-matured dc maintained a high il- release as compared to immature dc, although the 30-kda protein showed only as a trend. when the ifn-γ/il- ratio was calculated the 38-kda antigen showed the strongest il- bias although no significance was achieved by this calculation. these data indicate that dcs matured with the 38-kda antigen and as a trend also with the 30-kda antigen, allow seb-specific t cell responses biased towards a th profile, similar to immature dcs. although immune responses tested at the level of dcs against intracellular mtb and many of their antigens are dominated by ifn-γ producing cd8+ killer t cell and th responses, also th responses have been observed after infection of human monocytes with bcg prior to differentiation into dcs. treatment of pbmcs with peptides derived from the regions of difference antigens and expressed by mtb but not bcg led to th cytokine production. moreover, the presence of regulatory t cells and th cells may be lined to infection susceptibility to mtb. in fact, patients suffering from diabetes mellitus are more susceptible to tuberculosis development and this could be linked to a th2-bias of their anti-mtb responses. we provide evidence for two novel mtb antigens that induce th responses in vitro in spite of their capacity to elicit only incomplete dc maturation.  here we showed that the 30-kda and 38-kda antigens as mtb components induce only a partial dc maturation leading to a th shift in t cell polarization assays. since efficient immune protection from intracellular bacteria such as mtb depends on ifn-γ dependent cd4+ th and cytotoxic cd8+ t cell responses, the observed th shift can be envisaged as an immune escape mechanism by mtb. thus, using the two 30-kda and 38-kda antigens as an mtb vaccines may lead to a similar th shift in vivo. therefore these findings may have implications for vaccination strategies against mtb or for the pathological outcomes observed in mtb infected individuals suffering also from other diseases such as diabetes. although mtb interferes with several innate immune receptors to raise an immune response it has developed several mechanisms of immune evasion strategies to prevent elimination. although infected individuals generate immune responses and most people can control the infection they never reach complete mtb elimination and microbial sterility. thus, in most cases mtb infections represent a fine balance between immune reaction and immune evasion. the latter mechanisms may also be envisaged to prevent immune pathology by too strong immune reactions. together this reflects the co-evolution strategy of mtb. to develop effective vaccines without strong side effects this fine balance and all participating mechanisms have to be considered. a large part of the immune evasion by mtb is based on the persistent or latent infection of macrophages and the tlr-2-dependent down-regulation of antigen presenting function by mtb in these macrophages. the emerging role of dcs as regulators of tuberculosis disease has been elucidated only during the last years by genome-wide transcriptome analyses. it is well possible that also the induction of suboptimal maturation of dcs reflects another mechanism of mtb to target antigen presenting cells. in our system we studied selective mtb antigens and found that the two antigens 30-kda and 38-kda of mtb may be involved in immune evasion at the level of dcs. the quality of dc manipulation by mtb appears to differ from what is known from macrophages. while for macrophages the mhc-dependent antigen presenting function seems to dominate our data point to an intact antigen presentation as observed in the allo-mlr and seb-priming, but a bias towards th immunity. these in vitro data require now further validation in vivo but may indicate that the 30-kda and 38-kda antigens represent suboptimal mtb vaccine candidates. 
here we tested the stimulatory capacity of selective mtb antigens on human monocyte-derived dendritic cell maturation and cytokine production. deviation of immune responses by pathogens towards a th profile is a common mechanism of immune evasion, typically leading to the persistence of the microbes. dc maturation markers cd cd and cd were readily upregulated by h37ra- and h37rv-associated antigens, the 30-kda and 38-kda mtb antigens only partially induced these markers. together our data indicate that 30-kda and 38-kda mtb antigens induced only partial dc maturation shifting immune responses towards a th profile. all mtb antigens induced variable levels of il- and low levels of il- there was no release of il-12p detectable. although 99% of individuals infected with mtb develop a cd4+ th and cd8+ t cell mediated immunity as measured by tuberculin skin test, this results only in partial protection and mtb vaccines are not effective. although the proliferation levels of primary t cell responses were comparable using all the differentially stimulated dc, the 30-kda and 38-kda antigens showed a bias towards il- secretion of polarized cd4+ t cells after secondary stimulation as compared to h37ra and h37rv preparations.
 mycobacterium tuberculosis infections are still a major cause of death among all infectious diseases. although 99% of individuals infected with mtb develop a cd4+ th and cd8+ t cell mediated immunity as measured by tuberculin skin test, this results only in partial protection and mtb vaccines are not effective. deviation of immune responses by pathogens towards a th profile is a common mechanism of immune evasion, typically leading to the persistence of the microbes.  here we tested the stimulatory capacity of selective mtb antigens on human monocyte-derived dendritic cell maturation and cytokine production. dc maturation markers cd cd and cd were readily upregulated by h37ra- and h37rv-associated antigens, the 30-kda and 38-kda mtb antigens only partially induced these markers. all mtb antigens induced variable levels of il- and low levels of il- there was no release of il-12p detectable. substantial il-12p production was restricted to lps or h37ra and h37rv preparations. although the proliferation levels of primary t cell responses were comparable using all the differentially stimulated dc, the 30-kda and 38-kda antigens showed a bias towards il- secretion of polarized cd4+ t cells after secondary stimulation as compared to h37ra and h37rv preparations.  together our data indicate that 30-kda and 38-kda mtb antigens induced only partial dc maturation shifting immune responses towards a th profile. dendritic cellsmycobacterium tuberculosist helper cell responses 