BACKGROUND
pneumocystis is a fungus that can cause severe pneumonia in immunosuppressed hosts, especially those with hiv infection, but that can also cause pulmonary infection that is cleared by a robust immune response in healthy hosts.
while antibody responses are an important component of these responses, cell-mediated responses appear critical to the successful control of pneumocystis infection  <cit> .
pneumocystis can infect a wide range of host species, and each host is infected by a genetically unique pneumocystis species.
serologic studies among humans suggest widespread exposure to pneumocystis at an early age  <cit> .
the most abundant surface protein is an ~ <dig> - <dig>  mw protein known as the major surface glycoprotein   <cit> .
msg appears to play a role in mediating adhesion to host cells, possibly through binding to fibronectin or vitronectin  <cit> .
msg is encoded by a family of approximately  <dig> to  <dig> genes which are related but distinct; each pneumocystis species examined to date encodes a similar multi-copy gene family  <cit> .
while multiple different msgs may be expressed in a heavily infected immunosuppressed animal  <cit> , a single organism can express only a single msg  <cit> .
pneumocystis has developed a mechanism whereby there is a single expression site for msg genes  <cit> ; the gene to be expressed must be placed downstream of and in-frame with the upstream conserved sequence , which serves as a leader for msg and encodes a signal peptide for trafficking to the endoplasmic reticulum.
the mechanism for switching of expressed genes is unknown but may involve gene conversion.
while msg may play a functional role in pneumocystis biology such as in facilitating adhesion, the presence of multiple msg variants strongly suggests that pneumocystis has developed a mechanism for antigenic variation, similar to other organisms such as trypanosomes or borrelia <cit> .
antigenic variation could potentially allow persistence of infection or facilitate re-infection, contributing to the ubiquitous nature of this organism.
although pneumocystis is a pathogen of immunosuppressed hosts, pneumocystis can infect and induce a brisk and effective immune response in immunocompetent hosts  <cit> .
antigenic variation presumably evolved to evade host responses in the latter, given that immunosuppressed hosts would likely rarely be encountered in nature.
antigenic variation in many pathogens is designed to avoid humoral immune responses  <cit> .
in pneumocystis, although antibodies play a role in protection, cellular immune responses, especially cd4+ t cell responses, are critical for clearing infection and providing protective immunity  <cit> .
we hypothesized that a primary benefit of maintaining a broad msg repertoire is to allow switching of the expressed msg variant to evade cellular immune responses, especially cd4+ t cell responses, given that pneumocystis is exclusively an extracellular pathogen.
the current study was undertaken to address this hypothesis by examining:  <dig>  cellular and humoral responses in mice to two recombinant msg variants following immunization with recombinant proteins or native pneumocystis antigens, as well as following natural infection; and  <dig>  cellular proliferative and humoral responses in humans to a recombinant msg variant.
RESULTS
in order to study immune responses in mice to individual msgs, we expressed  <dig> recombinant msg variants,  <dig> and  <dig>  which differed from each other by 11%, in e. coli.
because of difficulties in expressing the entire msg as a single protein, we expressed each variant in two overlapping fragments, corresponding approximately to the amino and carboxyl halves of the full-length protein.
in addition, we expressed each fragment in multiple vectors, one of which was used for immunization studies, and another for in vitro assays.
this eliminated cross-reactive immune responses to the vector-encoded portion of the recombinant protein which we identified in preliminary studies.
we utilized proliferation to examine cellular t cell responses to pneumocystis antigens; this will primarily represent cd4+ t cell responses to protein  antigens.
in a subset of animals we also examined culture supernatant cytokine levels following antigenic stimulation.
attempts to detect intracellular cytokine responses, including interferon-gamma, interleukin- <dig>  interleukin- <dig>  and tumor necrosis factor-alpha, by flow cytometry were unsuccessful despite numerous attempts using a variety of pneumocystis antigens.
we initially immunized separate sets of  <dig> mice  <dig>   <dig>  or  <dig> times with the amino fragment of each of the two recombinant msg variants  <dig> and  <dig> , because there was less conservation in the amino  compared to the carboxyl  fragments.
we examined proliferative and antibody responses to both variants; results are shown in figure  <dig>  splenocytes from one of  <dig> mice immunized with a single dose of msg119am proliferated in response only to the immunizing antigen.
no proliferation was seen to either the immunizing or the non-immunizing variant fragment following  <dig> immunizations with either amino fragment.
following the 4th immunization, proliferative responses to the immunizing antigen were seen in splenocytes from  <dig> of  <dig> animals, while proliferation to the non-immunizing msg amino fragment was seen in splenocytes from  <dig> of these  <dig> animals.
in contrast, antibody responses as measured by elisa were seen in  <dig> of the  <dig> animals to both fragments following the first immunization, and in  <dig> of  <dig> animals to both fragments following  <dig> or  <dig> immunizations; optical densities were similar to both fragments regardless of whether  <dig> or  <dig> was used for immunization.
antibody responses to crude p. murina antigens were seen only in animals with antibody responses to the msg variants .
thus, cross-reactive proliferative responses were seen only following multiple immunizations, while cross-reactive antibody responses were seen after a single immunization.
no immune responses were seen to the recombinant msg fragments or crude p. murina antigens in control animals immunized with adjuvant alone .
to verify that the antibody responses to recombinant proteins recognized native msg, we utilized western blots with either crude pneumocystis antigens or purified native msg as the antigen.
using sera from animals immunized with the amino recombinant fragments, we were able to demonstrate reactivity with msg in both antigen preparations in  <dig> of  <dig> animals tested.
figure  <dig> shows results using sera from animals immunized with 107am or 119am.
we next examined proliferative and antibody responses following immunization with crude p. murina antigens.
msg is the most abundant protein in pneumocystis extracts, and in immunosuppressed animals, which are the source of this pneumocystis, multiple msg variants are expressed  <cit> .
splenocytes from immunized animals  did not proliferate in response to challenge with either the amino or the carboxyl fragment of either msg <dig> or msg <dig>  but did proliferate  to crude p. murina antigens, which were used to immunize the animals .
proliferative responses were also seen to purified native msg in splenocytes from immunized animals  but not in a control animal.
in contrast to this, antibody responses were seen to both amino  and carboxyl fragments of msg <dig> and msg <dig>  as well as to crude p. murina antigens, in all  <dig> animals.
antibody responses were also seen to purified native msg .
it is noteworthy that the antibody responses were greater  to both carboxyl fragments, which are more highly conserved, than to the amino fragments.
the antibody responses demonstrate that the animals were exposed to, and were able to mount an immune response to, msg in the crude antigen preparations.
because immunization with either recombinant or crude antigen preparations presents an artificial encounter with pneumocystis antigens, we wanted to examine immune responses following natural infection.
since immunosuppressed animals have inadequate immune responses to pneumocystis, which allow the organism to replicate in an uncontrolled or poorly controlled manner, we utilized a model in which healthy animals develop pneumocystis infection following exposure to an already infected seeder animal.
in this model, infection peaks at ~5-6 weeks following exposure, and is typically cleared by 10 weeks  <cit> .
splenocytes from exposed mice proliferated in response to crude pneumocystis antigens in  <dig> of  <dig> animals and to native msg in  <dig> of  <dig> animals, but none of the animals showed proliferation to any of the  <dig> recombinant msg protein fragments .
antibody responses were again seen to the carboxyl recombinant msg fragments  as well as to crude pneumocystis antigen and native msg , but not to the amino fragments , again indicating that these naturally infected animals were exposed to msg.
as an additional marker of cd <dig> responses, we examined in vitro cytokine production.
among the cytokines examined, il- <dig> and mcp- <dig> were consistently secreted by splenocytes from animals infected with p. murina when cultured with p. murina ag or native msg antigen, but not with the recombinant msg variants .
these responses represented memory responses, as no cytokine production was seen in cells from uninfected control animals cultured with the same antigens.
among the other cytokines, results with crude antigen or native msg were variably positive  but in no case were they produced in response to the recombinant antigens.
serologic studies have found that most humans have been exposed to pneumocystis at a young age and have detectable antibodies to msg.
to see if pbmcs from healthy humans can proliferate to recombinant msg, cells from  <dig> volunteers were cultured with humsg14am, humsg14ca, concanavalin a or tetanus toxoid.
no patient demonstrated proliferation to either humsg fragment after 5 days, while all samples proliferated in response to concanavalin a or tetanus toxoid.
moreover, sera from  <dig> of the  <dig> volunteers were positive in an elisa utilizing humsg14ca, while the 5th volunteer had high non-specific reactivity.
discussion
this study has demonstrated that while cross-reactive proliferative responses to two distinct recombinant msg variants were induced by multiple immunizations with recombinant msg protein, proliferative responses to these recombinant proteins were not seen following immunization with crude pneumocystis antigens, which contain msg, or following natural infection in a healthy host.
further more, these proteins did not induce il- <dig> and mcp- <dig> production in infected animals, unlike the crude antigen or purified native msg.
in contrast, nearly all animals developed antibody responses to the recombinant msg fragments, indicating that they recognized conserved b cell epitopes in msg.
since the cell-mediated  responses to extracellular proteins represent primarily cd4+ t cell responses, these data are thus consistent with the hypothesis that msg variants exhibit antigenic variation that evades cd4-mediated t cell responses rather than b cell responses.
because in vitro culture and genetic manipulation of pneumocystis is not currently possible, more direct methods for addressing this question, such as utilization of organisms that express a single msg gene, are currently not feasible, and thus indirect methods, as in the present study, must be utilized.
cd <dig> cells are critical to controlling infection with pneumocystis, and cd <dig> responses have previously been shown to target msg.
further, immunization with msg has been shown to induce proliferative responses  <cit>  but such immunization did not provide complete protection against pneumocystis infection  <cit> .
western blot studies demonstrated that the antibodies generated following immunization with recombinant proteins recognized native msg that was purified or present in crude antigen extracts.
thus conformational differences or differences in post-translational modifications that may exist between native and recombinant proteins did not prevent development of cross-reactive antibodies.
while t cell responses were not detected, this cannot be due to conformational differences since both cd <dig> and cd <dig> cells recognize processed peptides of ~8- <dig> amino acids that are not dependent on conformation.
we initially examined immune responses following immunization with recombinant msg variants, and found cross-reactivity in both proliferation and antibody responses after  <dig> immunizations, but only antibody responses following a lower exposure .
these initial studies also demonstrated that we could detect in vitro proliferation to the recombinant msg proteins.
of note, the animal that showed proliferation after only  <dig> immunization proliferated only to the immunizing msg variant, but developed antibodies that reacted with both msg variants.
no animals showed proliferative responses in the absence of antibody responses.
these data suggest that cd <dig> responses, as measured by proliferation, require repeat or prolonged exposure to msg.
such repeated exposure may allow for the expansion of a small number of initially responsive t cells or recruitment of new t cells recognizing additional msg epitopes.
interestingly, animals immunized with crude p. murina antigens demonstrated proliferative responses to the immunizing antigen and to purified native msg, but not to either of the recombinant msg variants, including the more conserved carboxyl portions .
a likely explanation for this observation is that the genes encoding msg <dig> and msg <dig> were not expressed by the p. murina organisms used for preparation of the crude antigen, since only  <dig> of the  <dig> to  <dig> msg genes is expressed by a p. murina organism at a time  <cit> .
while some of these cellular responses may also be to other p. murina antigens, possibly kexin or other unidentified p. murina antigens  <cit> , proliferation following exposure to native msg clearly demonstrates that cellular responses were directed in part against msg.
although the number of animals studied using native msg was small, precluding statistical analysis, the results were consistent with and supportive of those using crude antigen.
finally, naturally infected animals showed production of il- <dig> and mcp- <dig> and proliferation to crude p. murina antigen and to native msg protein but again lacked cross-reactive proliferative responses to either the amino or carboxyl portions of msg variants.
antibody responses were seen to the more conserved carboxyl fragment in most animals.
because we are not able to identify msg variants that are expressed by pneumocystis in individual naturally infected immunocompetent animals, we could not directly examine proliferative responses to the msg variants that were expressed by the infecting organism, nor could we compare their sequences to those of the recombinant msgs that were utilized in the in vitro assays.
our results are in contrast to those of theus et al.
<cit>  who demonstrated proliferative responses to a recombinant msg protein as well as to native msg, using splenocytes and t cell clones from naturally infected rats.
it is possible that the recombinant protein used in those studies was identical to or more closely related to the msg variants expressed by pneumocystis during infection of those animals.
interestingly, immunization with native or recombinant msg in rats provided partial protection from pneumocystis pneumonia, leading to a lower organism burden, while immunization with native msg in mice was not protective, though antibody responses were induced in both studies  <cit> .
we attempted to detect antigen-specific responses by intracellular cytokine staining using pneumocystis antigens that were positive in the proliferation assays, but were unable to detect such responses despite utilization of a variety of assay conditions.
we are aware of no publication to date that has successfully identified pneumocystis-specific responses using this assay.
this may be in part a function of the host-specific response to pneumocystis, since such responses have been easily identified for many other pathogens  <cit> .
serologic studies have demonstrated that most humans are exposed to pneumocystis at an early age  <cit> ; consistent with this, most humans, including those included in the current study, have antibodies to msg.
while we were not able to perform the same spectrum of studies in humans as in mice, we were able to demonstrate, in previously infected humans , absence of proliferative responses to a recombinant msg to which they maintained antibody responses.
although the total number of patients studied is small, and while it’s possible that the proliferative t cell responses have waned over time, when combined with the animal data these results support the concept that antigenic variation helps avoid t cell, but not b cell, mediated immune responses.
very few studies have examined immune responses to pneumocystis-specific antigens in humans, and all published studies have utilized rodent-derived antigens, which raises concerns that detected responses are non-specific, targeting xenogeneic, host-derived antigens, rather than being pneumocystis-specific  <cit> .
additional studies such as ours, that utilize purified or recombinant p. jirovecii-derived antigens, are clearly needed to better understand the interaction between pneumocystis and humans.
CONCLUSIONS
this study has shown that in mice, cellular responses  and antibody responses to variants of the msg of pneumocystis are discordant.
cross-reactive antibody responses occur following immunization with recombinant or native antigens, as well as following natural exposure to pneumocystis, while cross-reactive cellular responses are not seen following immunization with native antigens or following natural exposure.
the pneumocystis genome contains multiple msg gene variants, only one of which is expressed in an organism, which thus confers upon pneumocystis the potential for antigenic variation to avoid host immune responses.
given that cross-reactive cellular responses were not seen following immunization with native antigens or following natural exposure, while cross-reactive antibody responses were seen in these same animals , our results support the concept that antigenic variation in pneumocystis has evolved to evade cellular rather than antibody responses.
this is consistent with the observation that pneumocystis pneumonia occurs almost exclusively in hosts with predominantly cellular immunodeficiencies, such as hiv infection, reflecting the importance of cellular immune mechanisms in control of this infection.

 pneumocystis is a fungus that can cause severe pneumonia in immunosuppressed hosts, especially those with hiv infection, but that can also cause pulmonary infection that is cleared by a robust immune response in healthy hosts. while antibody responses are an important component of these responses, cell-mediated responses appear critical to the successful control of pneumocystis infection. pneumocystis can infect a wide range of host species, and each host is infected by a genetically unique pneumocystis species. serologic studies among humans suggest widespread exposure to pneumocystis at an early age. the most abundant surface protein is an ~ -  mw protein known as the major surface glycoprotein. msg appears to play a role in mediating adhesion to host cells, possibly through binding to fibronectin or vitronectin. msg is encoded by a family of approximately to genes which are related but distinct; each pneumocystis species examined to date encodes a similar multi-copy gene family. while multiple different msgs may be expressed in a heavily infected immunosuppressed animal, a single organism can express only a single msg. pneumocystis has developed a mechanism whereby there is a single expression site for msg genes; the gene to be expressed must be placed downstream of and in-frame with the upstream conserved sequence, which serves as a leader for msg and encodes a signal peptide for trafficking to the endoplasmic reticulum. the mechanism for switching of expressed genes is unknown but may involve gene conversion. while msg may play a functional role in pneumocystis biology such as in facilitating adhesion, the presence of multiple msg variants strongly suggests that pneumocystis has developed a mechanism for antigenic variation, similar to other organisms such as trypanosomes or borrelia. antigenic variation could potentially allow persistence of infection or facilitate re-infection, contributing to the ubiquitous nature of this organism. although pneumocystis is a pathogen of immunosuppressed hosts, pneumocystis can infect and induce a brisk and effective immune response in immunocompetent hosts. antigenic variation presumably evolved to evade host responses in the latter, given that immunosuppressed hosts would likely rarely be encountered in nature. antigenic variation in many pathogens is designed to avoid humoral immune responses. in pneumocystis, although antibodies play a role in protection, cellular immune responses, especially cd4+ t cell responses, are critical for clearing infection and providing protective immunity. we hypothesized that a primary benefit of maintaining a broad msg repertoire is to allow switching of the expressed msg variant to evade cellular immune responses, especially cd4+ t cell responses, given that pneumocystis is exclusively an extracellular pathogen. the current study was undertaken to address this hypothesis by examining: cellular and humoral responses in mice to two recombinant msg variants following immunization with recombinant proteins or native pneumocystis antigens, as well as following natural infection; and cellular proliferative and humoral responses in humans to a recombinant msg variant.  in order to study immune responses in mice to individual msgs, we expressed recombinant msg variants, and which differed from each other by 11%, in e. coli. because of difficulties in expressing the entire msg as a single protein, we expressed each variant in two overlapping fragments, corresponding approximately to the amino and carboxyl halves of the full-length protein. in addition, we expressed each fragment in multiple vectors, one of which was used for immunization studies, and another for in vitro assays. this eliminated cross-reactive immune responses to the vector-encoded portion of the recombinant protein which we identified in preliminary studies. we utilized proliferation to examine cellular t cell responses to pneumocystis antigens; this will primarily represent cd4+ t cell responses to protein antigens. in a subset of animals we also examined culture supernatant cytokine levels following antigenic stimulation. attempts to detect intracellular cytokine responses, including interferon-gamma, interleukin- interleukin- and tumor necrosis factor-alpha, by flow cytometry were unsuccessful despite numerous attempts using a variety of pneumocystis antigens. we initially immunized separate sets of mice or times with the amino fragment of each of the two recombinant msg variants and, because there was less conservation in the amino compared to the carboxyl fragments. we examined proliferative and antibody responses to both variants; results are shown in figure splenocytes from one of mice immunized with a single dose of msg119am proliferated in response only to the immunizing antigen. no proliferation was seen to either the immunizing or the non-immunizing variant fragment following immunizations with either amino fragment. following the 4th immunization, proliferative responses to the immunizing antigen were seen in splenocytes from of animals, while proliferation to the non-immunizing msg amino fragment was seen in splenocytes from of these animals. in contrast, antibody responses as measured by elisa were seen in of the animals to both fragments following the first immunization, and in of animals to both fragments following or immunizations; optical densities were similar to both fragments regardless of whether or was used for immunization. antibody responses to crude p. murina antigens were seen only in animals with antibody responses to the msg variants. thus, cross-reactive proliferative responses were seen only following multiple immunizations, while cross-reactive antibody responses were seen after a single immunization. no immune responses were seen to the recombinant msg fragments or crude p. murina antigens in control animals immunized with adjuvant alone. to verify that the antibody responses to recombinant proteins recognized native msg, we utilized western blots with either crude pneumocystis antigens or purified native msg as the antigen. using sera from animals immunized with the amino recombinant fragments, we were able to demonstrate reactivity with msg in both antigen preparations in of animals tested. figure shows results using sera from animals immunized with 107am or 119am. we next examined proliferative and antibody responses following immunization with crude p. murina antigens. msg is the most abundant protein in pneumocystis extracts, and in immunosuppressed animals, which are the source of this pneumocystis, multiple msg variants are expressed. splenocytes from immunized animals did not proliferate in response to challenge with either the amino or the carboxyl fragment of either msg or msg but did proliferate to crude p. murina antigens, which were used to immunize the animals. proliferative responses were also seen to purified native msg in splenocytes from immunized animals but not in a control animal. in contrast to this, antibody responses were seen to both amino and carboxyl fragments of msg and msg as well as to crude p. murina antigens, in all animals. antibody responses were also seen to purified native msg. it is noteworthy that the antibody responses were greater to both carboxyl fragments, which are more highly conserved, than to the amino fragments. the antibody responses demonstrate that the animals were exposed to, and were able to mount an immune response to, msg in the crude antigen preparations. because immunization with either recombinant or crude antigen preparations presents an artificial encounter with pneumocystis antigens, we wanted to examine immune responses following natural infection. since immunosuppressed animals have inadequate immune responses to pneumocystis, which allow the organism to replicate in an uncontrolled or poorly controlled manner, we utilized a model in which healthy animals develop pneumocystis infection following exposure to an already infected seeder animal. in this model, infection peaks at ~5-6 weeks following exposure, and is typically cleared by 10 weeks. splenocytes from exposed mice proliferated in response to crude pneumocystis antigens in of animals and to native msg in of animals, but none of the animals showed proliferation to any of the recombinant msg protein fragments. antibody responses were again seen to the carboxyl recombinant msg fragments as well as to crude pneumocystis antigen and native msg, but not to the amino fragments, again indicating that these naturally infected animals were exposed to msg. as an additional marker of cd responses, we examined in vitro cytokine production. among the cytokines examined, il- and mcp- were consistently secreted by splenocytes from animals infected with p. murina when cultured with p. murina ag or native msg antigen, but not with the recombinant msg variants. these responses represented memory responses, as no cytokine production was seen in cells from uninfected control animals cultured with the same antigens. among the other cytokines, results with crude antigen or native msg were variably positive but in no case were they produced in response to the recombinant antigens. serologic studies have found that most humans have been exposed to pneumocystis at a young age and have detectable antibodies to msg. to see if pbmcs from healthy humans can proliferate to recombinant msg, cells from volunteers were cultured with humsg14am, humsg14ca, concanavalin a or tetanus toxoid. no patient demonstrated proliferation to either humsg fragment after 5 days, while all samples proliferated in response to concanavalin a or tetanus toxoid. moreover, sera from of the volunteers were positive in an elisa utilizing humsg14ca, while the 5th volunteer had high non-specific reactivity. discussion this study has demonstrated that while cross-reactive proliferative responses to two distinct recombinant msg variants were induced by multiple immunizations with recombinant msg protein, proliferative responses to these recombinant proteins were not seen following immunization with crude pneumocystis antigens, which contain msg, or following natural infection in a healthy host. further more, these proteins did not induce il- and mcp- production in infected animals, unlike the crude antigen or purified native msg. in contrast, nearly all animals developed antibody responses to the recombinant msg fragments, indicating that they recognized conserved b cell epitopes in msg. since the cell-mediated responses to extracellular proteins represent primarily cd4+ t cell responses, these data are thus consistent with the hypothesis that msg variants exhibit antigenic variation that evades cd4-mediated t cell responses rather than b cell responses. because in vitro culture and genetic manipulation of pneumocystis is not currently possible, more direct methods for addressing this question, such as utilization of organisms that express a single msg gene, are currently not feasible, and thus indirect methods, as in the present study, must be utilized. cd cells are critical to controlling infection with pneumocystis, and cd responses have previously been shown to target msg. further, immunization with msg has been shown to induce proliferative responses but such immunization did not provide complete protection against pneumocystis infection. western blot studies demonstrated that the antibodies generated following immunization with recombinant proteins recognized native msg that was purified or present in crude antigen extracts. thus conformational differences or differences in post-translational modifications that may exist between native and recombinant proteins did not prevent development of cross-reactive antibodies. while t cell responses were not detected, this cannot be due to conformational differences since both cd and cd cells recognize processed peptides of ~8- amino acids that are not dependent on conformation. we initially examined immune responses following immunization with recombinant msg variants, and found cross-reactivity in both proliferation and antibody responses after immunizations, but only antibody responses following a lower exposure. these initial studies also demonstrated that we could detect in vitro proliferation to the recombinant msg proteins. of note, the animal that showed proliferation after only immunization proliferated only to the immunizing msg variant, but developed antibodies that reacted with both msg variants. no animals showed proliferative responses in the absence of antibody responses. these data suggest that cd responses, as measured by proliferation, require repeat or prolonged exposure to msg. such repeated exposure may allow for the expansion of a small number of initially responsive t cells or recruitment of new t cells recognizing additional msg epitopes. interestingly, animals immunized with crude p. murina antigens demonstrated proliferative responses to the immunizing antigen and to purified native msg, but not to either of the recombinant msg variants, including the more conserved carboxyl portions. a likely explanation for this observation is that the genes encoding msg and msg were not expressed by the p. murina organisms used for preparation of the crude antigen, since only of the to msg genes is expressed by a p. murina organism at a time. while some of these cellular responses may also be to other p. murina antigens, possibly kexin or other unidentified p. murina antigens, proliferation following exposure to native msg clearly demonstrates that cellular responses were directed in part against msg. although the number of animals studied using native msg was small, precluding statistical analysis, the results were consistent with and supportive of those using crude antigen. finally, naturally infected animals showed production of il- and mcp- and proliferation to crude p. murina antigen and to native msg protein but again lacked cross-reactive proliferative responses to either the amino or carboxyl portions of msg variants. antibody responses were seen to the more conserved carboxyl fragment in most animals. because we are not able to identify msg variants that are expressed by pneumocystis in individual naturally infected immunocompetent animals, we could not directly examine proliferative responses to the msg variants that were expressed by the infecting organism, nor could we compare their sequences to those of the recombinant msgs that were utilized in the in vitro assays. our results are in contrast to those of theus et al. who demonstrated proliferative responses to a recombinant msg protein as well as to native msg, using splenocytes and t cell clones from naturally infected rats. it is possible that the recombinant protein used in those studies was identical to or more closely related to the msg variants expressed by pneumocystis during infection of those animals. interestingly, immunization with native or recombinant msg in rats provided partial protection from pneumocystis pneumonia, leading to a lower organism burden, while immunization with native msg in mice was not protective, though antibody responses were induced in both studies. we attempted to detect antigen-specific responses by intracellular cytokine staining using pneumocystis antigens that were positive in the proliferation assays, but were unable to detect such responses despite utilization of a variety of assay conditions. we are aware of no publication to date that has successfully identified pneumocystis-specific responses using this assay. this may be in part a function of the host-specific response to pneumocystis, since such responses have been easily identified for many other pathogens. serologic studies have demonstrated that most humans are exposed to pneumocystis at an early age; consistent with this, most humans, including those included in the current study, have antibodies to msg. while we were not able to perform the same spectrum of studies in humans as in mice, we were able to demonstrate, in previously infected humans, absence of proliferative responses to a recombinant msg to which they maintained antibody responses. although the total number of patients studied is small, and while it’s possible that the proliferative t cell responses have waned over time, when combined with the animal data these results support the concept that antigenic variation helps avoid t cell, but not b cell, mediated immune responses. very few studies have examined immune responses to pneumocystis-specific antigens in humans, and all published studies have utilized rodent-derived antigens, which raises concerns that detected responses are non-specific, targeting xenogeneic, host-derived antigens, rather than being pneumocystis-specific. additional studies such as ours, that utilize purified or recombinant p. jirovecii-derived antigens, are clearly needed to better understand the interaction between pneumocystis and humans.  this study has shown that in mice, cellular responses and antibody responses to variants of the msg of pneumocystis are discordant. cross-reactive antibody responses occur following immunization with recombinant or native antigens, as well as following natural exposure to pneumocystis, while cross-reactive cellular responses are not seen following immunization with native antigens or following natural exposure. the pneumocystis genome contains multiple msg gene variants, only one of which is expressed in an organism, which thus confers upon pneumocystis the potential for antigenic variation to avoid host immune responses. given that cross-reactive cellular responses were not seen following immunization with native antigens or following natural exposure, while cross-reactive antibody responses were seen in these same animals, our results support the concept that antigenic variation in pneumocystis has evolved to evade cellular rather than antibody responses. this is consistent with the observation that pneumocystis pneumonia occurs almost exclusively in hosts with predominantly cellular immunodeficiencies, such as hiv infection, reflecting the importance of cellular immune mechanisms in control of this infection. 
proliferative responses to each of two recombinant msg variant proteins were seen in mice immunized with either recombinant protein, but no proliferation to these antigens was seen in mice immunized with crude pneumocystis antigens or in mice that had cleared infection, although the latter animals demonstrated proliferative responses to crude pneumocystis antigens and native msg. cross-reactive antibody responses to msg variants are common, while cross-reactive t cell responses are uncommon; these results support the hypothesis that pneumocystis utilizes switching of msg variant expression to avoid host t cell responses. we examined splenic t cell proliferative and cytokine as well as serum antibody responses to recombinant and native pneumocystis antigens in immunized or pneumocystis-infected mice. antibody responses to the recombinant p. murina msg variant proteins were seen in all groups of animals, demonstrating that all groups were exposed to and mounted immune responses to msg. no human pbmc samples proliferated following stimulation with p. jirovecii msg, while antibody responses were detected in sera from of samples. il- and mcp- were produced in previously infected animals in response to the same antigens, but not to recombinant antigens. in addition, immune responses were examined in healthy humans.
 the major surface glycoprotein of pneumocystis is encoded by approximately to unique but related genes. msg diversity may represent a mechanism for immune escape from host t cell responses. we examined splenic t cell proliferative and cytokine as well as serum antibody responses to recombinant and native pneumocystis antigens in immunized or pneumocystis-infected mice. in addition, immune responses were examined in healthy humans.  proliferative responses to each of two recombinant msg variant proteins were seen in mice immunized with either recombinant protein, but no proliferation to these antigens was seen in mice immunized with crude pneumocystis antigens or in mice that had cleared infection, although the latter animals demonstrated proliferative responses to crude pneumocystis antigens and native msg. il- and mcp- were produced in previously infected animals in response to the same antigens, but not to recombinant antigens. antibody responses to the recombinant p. murina msg variant proteins were seen in all groups of animals, demonstrating that all groups were exposed to and mounted immune responses to msg. no human pbmc samples proliferated following stimulation with p. jirovecii msg, while antibody responses were detected in sera from of samples.  cross-reactive antibody responses to msg variants are common, while cross-reactive t cell responses are uncommon; these results support the hypothesis that pneumocystis utilizes switching of msg variant expression to avoid host t cell responses. antigenic variationimmune responsemajor surface glycoproteinpneumocystis 