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Research in our lab focuses on the complex immunobiological relationship between host and parasite in African trypanosomiasis, a fatal protozoan disease of man and animals. Our studies examine the immunogenetics of resistance to disease by utilizing genetically altered host animals and genetically modified trypanosomes in order to dissect out those biological elements that are functionally and genetically linked to resistance. We also examine programmed changes in parasite gene and protein expression that appear to be linked to changes in virulence. Genes and proteins differentially expressed between genetically related high and low virulence trypanosomes have been isolated and characterized, and candidate virulence genes are being modified in order to address the subcellular basis for virulence in African trypanosomiasis. In summary, research in the Mansfield lab elucidates centrally important host and parasite biological events that determine the course of infection, and our future studies are aimed at modifying these events in order to control or cure trypanosomiasis.
Microbiology 375: Fundamentals of Immunology (As Informed by Microbial Pathogens)
Microbiology 790: Immunology of Infectious Disease
Microbiology 731: MDTP Student Seminar
Microbial Pathogenesis and Host Response Group
Cell and Molecular Parasitology Training Program
MBTG Training Program
Executive Joint Professor of Medical Microbiology and Immunology
Board of Directors, Tandem Press
No abstract available.
African trypanosomes cause human and animal African trypanosomiases, which are chronic, debilitating and often fatal diseases of people and livestock in sub-Saharan Africa. The extracellular protozoan parasites are exemplars of antigenic variation. They direct host-protective B-cell and T-cell immune responses towards hypervariable components of their variable surface glycoprotein coat and evade immune elimination by generating new surface coat antigenic variants at a rate that supersedes immune destruction. This results in recurring waves of parasitemia, tissue invasion and escalating immunopathology in trypanosomiasis-susceptible hosts. Here, we discuss the possibility that host control of African trypanosomes might be improved by immunization with conserved VSG peptides and invariant surface glycoproteins. Infection-induced T-cell recall responses to these typically poorly expressed or nonimmunogenic parasite components induce tissue phagocytes to produce microbicidal materials that kill trypanosomes. Preliminary data that support this immune-enhancing vaccine strategy are discussed, as are host and parasite interactions that might downregulate the protective responses. These include infection-induced immunosuppression and increasing virulence of infecting parasites over time.
The experimental studies of Brucei group trypanosomes presented here demonstrate that the balance of host and parasite factors, especially IFN-γ GPI-sVSG respectively, and the timing of cellular exposure to them, dictate the predominant MP and DC activation profiles present at any given time during infection and within specific tissues. The timing of changes in innate immune cell functions following infection consistently support the conclusion that the key events controlling host resistance occur within a short time following initial exposure to the parasite GPI substituents. Once the changes in MP and DC activities are initiated, there appears little that the host can do to reverse these changes and alter the final outcome of these regulatory events. Instead, despite the availability of multiple innate and adaptive immune mechanisms that can control parasites, there is an inability to control trypanosome numbers sufficiently to prevent the emergence and establishment of virulent trypanosomes that eventually kill the host. Overall it appears that trypanosomes have carefully orchestrated the host innate and adaptive immune response so that parasite survival and transmission, and alterations of host immunity, are to its ultimate benefit.
The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.
Th1 cell responses to the variant surface glycoprotein (VSG) of African trypanosomes play a critical role in controlling infection through the production of IFN-gamma, but the role of APCs in the induction and regulation of T cell-mediated protection is poorly understood. In this study, we have investigated the Ag presentation capabilities of dendritic cells (DCs) and macrophages during early trypanosome infection in relatively resistant responder and susceptible nonresponder mouse strains. Splenic DCs appeared to be the primary cell responsible for activating naive VSG-specific Th cell responses in resistant responder animals through the coordinated up-regulation of costimulatory molecules, secretion of IL-12, and presentation of VSG peptides to T cells in vivo. Splenic DC depletion and the down-regulation of costimulatory markers on splenic macrophages were observed in susceptible animals and may be associated with the inability of these animals to elicit a significant VSG-specific T cell response. In contrast to splenic APCs, peritoneal macrophages secreted NO, failed to activate naive Th cells in vitro, and presented relatively low levels of VSG peptides to T cells in vivo. Thus, VSG-specific Th1 cell responses may be determined by tissue- and cell-specific differences in Ag presentation. Additionally, all APCs from resistant and susceptible strains displayed a reduced ability to process and present newly encountered exogenous Ag, including new VSG molecules, during high parasitemia. Thus, initial uptake of VSG (or other trypanosome factors) may interfere with Ag presentation and have dramatic consequences for subsequent T cell responses to other proteins.
Variable subregions within the variant surface glycoprotein (VSG) coat displayed by African trypanosomes are predicted sites for T- and B-cell recognition. Hypervariable subregion 1 (HV-1) is localized to an internal amphipathic alpha helix in VSG monomers and may have evolved due to selective pressure by host T-cell responses to epitopes within this subregion. The prediction of T-cell receptor-reactive sites and major histocompatibility complex class II binding motifs within the HV-1 subregion, coupled with the conservation of amino acid residues in other regions of the molecule sufficient to maintain secondary and tertiary VSG structure, prompted us to test the hypothesis that Th cells may preferentially recognize HV-1 subregion peptides. Thus, we examined the fine specificity of VSG-specific T-cell lines, T-cell hybridomas, and Th cells activated during infection. Our results demonstrate that T-cell epitopes are distributed throughout the N-terminal domain of VSG but are not clustered exclusively within HV-1 or other hypervariable subregions. In contrast, T-cell-reactive sites were not detected within the relatively conserved C-terminal domain of VSG. Overall, this study is the first to dissect the fine specificity of T-cell responses to the trypanosome VSG and suggests that evolution of a conserved HV-1 region may be unrelated to selective pressures exerted by host T-cell responses. This study also demonstrates that T cells do not recognize the relatively invariant C-terminal region of the VSG molecule during infection, suggesting that it could serve as a potential subunit vaccine to provide variant cross-specific immunity for African trypanosomiasis.
Variable subregions within the variant surface glycoprotein (VSG) coat displayed by African trypanosomes are predicted sites for T- and B-cell recognition. Hypervariable subregion 1 (HV-1) is localized to an internal amphipathic alpha helix in VSG monomers and may have evolved due to selective pressure by host T-cell responses to epitopes within this subregion. The prediction of T-cell receptor-reactive sites and major histocompatibility complex class II binding motifs within the HV-1 subregion, coupled with the conservation of amino acid residues in other regions of the molecule sufficient to maintain secondary and tertiary VSG structure, prompted us to test the hypothesis that Th cells may preferentially recognize HV-1 subregion peptides. Thus, we examined the fine specificity of VSG-specific T-cell lines, T-cell hybridomas, and Th cells activated during infection. Our results demonstrate that T-cell epitopes are distributed throughout the N-terminal domain of VSG but are not clustered exclusively within HV-1 or other hypervariable subregions. In contrast, T-cell-reactive sites were not detected within the relatively conserved C-terminal domain of VSG. Overall, this study is the first to dissect the fine specificity of T-cell responses to the trypanosome VSG and suggests that evolution of a conserved HV-1 region may be unrelated to selective pressures exerted by host T-cell responses. This study also demonstrates that T cells do not recognize the relatively invariant C-terminal region of the VSG molecule during infection, suggesting that it could serve as a potential subunit vaccine to provide variant cross-specific immunity for African trypanosomiasis.
Macrophages express a spectrum of proinflammatory and regulatory mediators during African trypanosomiasis. Microarray analyses revealed similar profiles of induced genes in macrophages stimulated with the trypanosome soluble variant surface glycoprotein in vitro and in macrophages taken from infected mice. Genes associated with the acute phase response and with type I IFN responses were prominent components of the macrophage activation profiles expressed within 72 h in vitro and in vivo. Thus, induction of proinflammatory gene expression is a characteristic of early trypanosome infection that is driven primarily by soluble variant surface glycoprotein exposure, and it may be that IFN-alpha/beta plays a central role in regulation of early resistance to trypanosomes. To test this hypothesis, we assessed parameters of infection in mouse strains with genetic alterations in the IFN-alpha/beta response pathway. We found that Ifnar1(-/-) mice, which lack the receptor for type I IFNs, exhibited delayed control of parasite burden during the first week of infection and died earlier than did wild-type controls. However, infection of Ubp43(-/-) mice, which are hyperresponsive to type I IFNs, did not exhibit enhanced resistance to trypanosomes. Instead, these animals also failed to control parasite burden and were more susceptible than wild-type animals. Additionally, the Ubp43(-/-) mice exhibited a significant defect in IFN-gamma production, which is definitively linked to host resistance in trypanosomiasis. These results show that type I IFNs play a role in early control of parasites in infected mice but may contribute to down-regulation of IFN-gamma production and subsequent loss of host resistance later in infection.
The variant surface glycoprotein (VSG) coat of African trypanosomes exhibits immunobiological functions distinct from its prominent role as a variant surface antigen. In order to address questions regarding immune stealth effects of VSG switch-variant coats, and the innate immune system activating effects of shed VSG substituents, several groups have genetically modified the ability of trypanosomes to express or release VSG during infection of the mammalian host. The role of mosaic surface coats expressed by VSG switch-variants (VSG double-expressors) in escaping early immune detection, and the role of VSG glycosylphosphatidylinositol (GPI) anchor substituents in regulating host immunity have been revealed, respectively, by stable co-expression of an exogenous VSG gene in trypanosomes expressing an endogenous VSG gene, and by knocking out the genetic locus for GPI-phospholipase C (PLC) that releases VSG from the membrane. Both approaches to genetic modification of African trypanosomes have suggested interesting and unexpected immunobiological effects associated with surface coat molecules.
PilE is the primary subunit of type IV pili from Neisseria gonorrhoeae and contains a surface-exposed hypervariable region thought to be one feature of pili that has prevented development of a pilin-based vaccine. We have created a three-dimensional structure-based antigen by replacing the hypervariable region of PilE with an aspartate-glutamine linker chosen from the sequence of Pseudomonas aeruginosa PilA. We then characterized murine immune responses to this novel protein to determine if conserved PilE regions could serve as a vaccine candidate. The control PilE protein elicited strong T-cell-dependent B-cell responses that are specific to epitopes in both the hypervariable deletion and control proteins. In contrast, the hypervariable deletion protein was unable to elicit an immune response in mice, suggesting that in the absence of the hypervariable region, the conserved regions of PilE alone are not sufficient for antibody production. Further analysis of these PilE proteins with suppressor cell assays showed that neither suppresses T- or B-cell responses, and flow cytometry experiments suggested that they do not exert suppressor effects by activating T regulatory cells. Our results show that in the murine model, the hypervariable region of PilE is required to activate immune responses to pilin, whereas the conserved regions are unusually nonimmunogenic. In addition, we show that both hypervariable and conserved regions of pilin are not suppressive, suggesting that PilE does not cause the decrease in T-cell populations observed during gonococcal cervicitis.
The GPI residues of soluble variant surface glycoprotein (sVSG) molecules released from the membrane of African trypanosomes during infection induce macrophage activation events. In this study, we demonstrate that the trypanosome sVSG molecule binds to the membrane of murine RAW 264.7 macrophages and activates the NF-kappaB cascade independently of a TLR-mediated interaction. The binding of fluorochrome-labeled sVSG molecules to macrophage membranes was saturable, was inhibited by the scavenger receptor-specific ligand maleylated BSA, and was followed by rapid intracellular uptake of the molecules and subsequent internalization to lysosomal compartments. Inhibition of cellular phagocytic and endocytic uptake processes by cytochalasin B and monodansylcadaverine, respectively, revealed that sVSG internalization was necessary for IkappaBalpha degradation and occurred by an actin-dependent, clathrin-independent process. Activation of RAW 264.7 cells by sVSG following treatment of the cells with the TRAF6 inhibitory peptide DIVK resulted in enhanced NF-kappaB signaling, suggesting both that TRAF6-dependent TLR activation of the pathway alone is not required for signaling and that TLR pathway components may negatively regulate expression of sVSG-induced signaling. These results demonstrate that stimulation of macrophages by sVSG involves a complex process of receptor-mediated binding and uptake steps, leading to both positive and negative signaling events that ultimately regulate cellular activation.
Relative resistance to African trypanosomiasis is based on the development of a type I cytokine response, which is partially dependent on innate immune responses generated through MyD88 and Toll-like receptor 9 (TLR9). Therefore, we asked whether enhancement of the immune response by artificial stimulation with CpG oligodeoxynucleotide (ODN), a TLR9 agonist, would result in enhanced protection against trypanosomes. In susceptible BALB/c mice, relative resistance to infection was significantly enhanced by CpG ODN treatment and was associated with decreased parasite burden, increased cytokine production, and elevated parasite-specific B- and T-cell responses. In relatively resistant C57BL/6 mice, survival was not enhanced but early parasitemia levels were reduced 100-fold and the majority of the parasites were nondividing, short stumpy (SS) forms. CpG ODN treatment of lymphocyte-deficient C57BL/6-scid and BALB/cByJ-scid mice also enhanced survival and reduced parasitemia, indicating that innate resistance to trypanosome infection can be enhanced. In C57BL/6-scid and BALB/cByJ-scid mice, the parasites were also predominantly SS forms during the outgrowth of parasitemia. However, the effect of CpG ODN treatment on parasite morphology was not as marked in gamma interferon (IFN-gamma)-knockout mice, suggesting that downstream effects of IFN-gamma production may play a discrete role in parasite cell differentiation. Overall, these studies provide the first evidence that enhancement of resistance to African trypanosomes can be induced in susceptible animals in a TLR9-dependent manner and that CpG ODN treatment may influence the developmental life cycle of the parasites.
Activation of a type I cytokine response is important for early resistance to infection with Trypanosoma brucei rhodesiense, the extracellular protozoan parasite that causes African sleeping sickness. The work presented here demonstrates that trypanosome DNA activates macrophages to produce factors that may contribute to this response. Initial results demonstrated that T. brucei rhodesiense DNA was present in the plasma of C57BL/6 and C57BL/6-scid mice following infection. Subsequently, the effect of trypanosome DNA on macrophages was investigated; parasite DNA was found to be less stimulatory than Escherichia coli DNA but more stimulatory than murine DNA, as predicted by the CG dinucleotide content. Trypanosome DNA stimulated the induction of a signal transduction cascade associated with Toll-like receptor signaling in RAW 264.7 macrophage cells. The signaling cascade led to expression of mRNAs, including interleukin-12 (IL-12) p40, IL-6, IL-10, cyclooxygenase-2, and beta interferon. The treatment of RAW 264.7 cells and bone marrow-derived macrophages with trypanosome DNA induced the production of NO, prostaglandin E2, and the cytokines IL-6, IL-10, IL-12, and tumor necrosis factor alpha. In all cases, DNase I treatment of T. brucei rhodesisense DNA abolished the activation. These results suggest that T. brucei rhodesiense DNA serves as a ligand for innate immune cells and may play an important contributory role in early stimulation of the host immune response during trypanosomiasis.
Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.
African trypanosomes are well known for their ability to avoid immune elimination by switching the immunodominant variant surface glycoprotein (VSG) coat during infection. However, antigenic variation is only one of several means by which trypanosomes manipulate the immune system of their hosts. In this article, the role of parasite factors such as GPI anchor residues of the shed VSG molecule and the release of CpG DNA, in addition to host factors such as IFN-gamma, in regulating key aspects of innate and acquired immunity during infection is examined. The biological relevance of these immunoregulatory events is discussed in the context of host and parasite survival.
Host resistance to African trypanosomiasis is partially dependent on an early and strong T-independent B-cell response against the variant surface glycoprotein (VSG) coat expressed by trypanosomes. The repetitive array of surface epitopes displayed by a monotypic surface coat, in which identical VSG molecules are closely packed together in a uniform architectural display, cross-links cognate B-cell receptors and initiates T-independent B-cell activation events. However, this repetitive array of identical VSG epitopes is altered during the process of antigenic variation, when former and nascent VSG proteins are transiently expressed together in a mosaic surface coat. Thus, T-independent B-cell recognition of the trypanosome surface coat may be disrupted by the introduction of heterologous VSG molecules into the coat structure. To address this hypothesis, we transformed Trypanosoma brucei rhodesiense LouTat 1 with the 117 VSG gene from Trypanosoma brucei brucei MiTat 1.4 in order to produce VSG double expressers; coexpression of the exogenous 117 gene along with the endogenous LouTat 1 VSG gene resulted in the display of a mosaic VSG coat. Results presented here demonstrate that the host's ability to produce VSG-specific antibodies and activate B cells during early infection with VSG double expressers is compromised relative to that during infection with the parental strain, which displays a monotypic coat. These findings suggest a previously unrecognized mechanism of immune response evasion in which coat-switching trypanosomes fail to directly activate B cells until coat VSG homogeneity is achieved. This process affords an immunological advantage to trypanosomes during the process of antigenic variation.
Macrophages are centrally involved in the host immune response to infection with Trypanosoma brucei rhodesiense, a protozoan parasite responsible for human sleeping sickness in Africa. During trypanosome infections, the host is exposed to parasite-derived molecules that mediate macrophage activation, specifically GPI anchor substituents associated with the shed variant surface glycoprotein (VSG), plus the host-activating agent IFN-gamma, which is derived from activated T cells and is essential for resistance to trypanosomes. In this study, we demonstrate that the level and timing of exposure of macrophages to IFN-gamma vs GPI ultimately determine the macrophage response at the level of induced gene expression. Treatment of macrophages with IFN-gamma followed by GIP-sVSG (the soluble form of VSG containing the glycosylinositolphosphate substituent that is released by parasites) stimulated the induction of gene expression, including transcription of TNF-alpha, IL-6, GM-CSF, and IL-12p40. In contrast, treatment of macrophages with GIP-sVSG before IFN-gamma stimulation resulted in a marked reduction of IFN-gamma-induced responses, including transcription of inducible NO synthase and secretion of NO. Additional experiments revealed that the inhibitory activity of GIP-sVSG was associated with reduction in the level of STAT1 phosphorylation, an event required for IFN-gamma-induced macrophage activation. These results suggest that modulation of specific aspects of the IFN-gamma response may be one mechanism by which trypanosomes overcome host resistance during African trypanosomiasis.
Resistance to African trypanosomes is dependent on B cell and Th1 cell responses to the variant surface glycoprotein (VSG). While B cell responses to VSG control levels of parasitemia, the cytokine responses of Th1 cells to VSG appear to be linked to the control of parasites in extravascular tissues. We have recently shown that IFN-gamma knockout (IFN-gamma KO) mice are highly susceptible to infection and have reduced levels of macrophage activation compared to the wild-type C57BL/6 (WT) parent strain, even though parasitemias were controlled by VSG-specific antibody responses in both strains. In the present work, we examine the role of IFN-gamma in the induction of nitric oxide (NO) production and host resistance and in the development of suppressor macrophage activity in mice infected with Trypanosoma brucei rhodesiense. In contrast to WT mice, susceptible IFN-gamma KO mice did not produce NO during infection and did not develop suppressor macrophage activity, suggesting that NO might be linked to resistance but that suppressor cell activity was not associated with resistance or susceptibility to trypanosome infection. To further examine the consequence of inducible NO production in infection, we monitored survival, parasitemia, and Th cell cytokine production in iNOS KO mice. While survival times and parasitemia of iNOS KO mice did not differ significantly from WT mice, VSG-specific Th1 cells from iNOS KO mice produced higher levels of IFN-gamma and IL-2 than cells from WT mice. Together, these results show for the first time that inducible NO production is not the central defect associated with susceptibility of IFN-gamma KO mice to African trypanosomes, that IFNgamma-induced factors other than iNOS may be important for resistance to the trypanosomes, and that suppressor macrophage activity is not linked to either the resistance or the susceptibility phenotypes.
The role of variant surface glycoprotein (VSG)-specific Th cell responses in determining resistance to the African trypanosomes was examined by comparing Th cell responses in relatively resistant and susceptible mice as well as in cytokine gene knockout mice infected with Trypanosoma brucei rhodesiense. Resistant B10.BR and C57BL/6 mice expressed Th1 cell cytokine responses to VSG stimulation during infection, while susceptible C3H mice produced weak or no Th1 cell cytokine responses. Neither resistant B10.BR and C57BL/6 mice nor susceptible C3H mice made detectable Th2 cell cytokine responses to parasite Ag. To more closely examine the potential role of IFN-gamma and other cytokines in host resistance, we determined the resistance phenotypes and Th cell responses of IFN-gamma and IL-4 knockout mice. Infected C57BL/6-IFN-gamma knockout mice were as susceptible as C57BL/6-scid mice and made an IL-2, but not an IL-4, cytokine response to VSG, while C57BL/6-IL-4 knockout mice were as resistant as the wild-type strain and exhibited both IL-2 and IFN-gamma cytokine responses. Passive transfer of spleen cells from wild-type mice to IFN-gamma knockout mice resulted in enhanced survival. Both wild-type and IFN-gamma knockout mice controlled parasitemia with VSG-specific Ab responses, although parasitemias were higher in the IFN-gamma knockout mice. Overall, this study demonstrates for the first time that relative resistance to African trypanosomes is associated with a strong Th1 cell response to parasite Ags, that IFN-gamma, but not IL-4, is linked to host resistance, and that susceptible animals do not make compensatory Th2 cell responses in the absence of Th1 cell cytokine responses.
The variant surface glycoprotein (VSG) gene of Trypanosoma brucei rhodesiense LouTat 1.5, a defined African trypanosome variant antigenic type, was cloned and sequenced. Southern blot analysis revealed 2 DNA restriction fragments in both VSG 1.5 expressor and nonexpressor populations, suggesting that there are 2 genomic copies of the VSG 1.5 gene and no expression-linked copies. Pulsed-field gel electrophoresis followed by Southern blot analysis showed that each copy of the VSG 1.5 gene exists on a separate large chromosome in both the expressor (approximately 3.5- and 4-megabase (Mb) chromosomes) and nonexpressor (approximately 4- and 5.7-Mb chromosomes) populations. Thus, VSG genes may be present on larger chromosomes than previously reported. Sequence analysis and alignments revealed that the VSG 1.5 molecule is a class B VSG with 12 cysteine residues in the N-terminus and is classified as a type 2 VSG based on C-terminus motifs. This classification shows that the VSG 1.5 molecule represents a relatively rare VSG class and type. Taken together, these studies provide additional information on VSG genes and proteins and supply the foundation for structure-function analysis of the VSG 1.5 surface antigen expressed by trypanosomes of the LouTat 1 serodeme.
This study examines B-cell immunoglobulin (Ig) class-switching events in the context of parasite antigen-specific Th-cell responses in experimental African trypanosomiasis. Inbred mice were infected with Trypanosoma brucei rhodesiense, and the coordinate stimulation of Th-cell cytokine responses and B-cell responses to the trypanosome variant surface glycoprotein (VSG) was measured. The cytokines produced by T cells in response to VSG, at both the transcript and protein levels, were gamma interferon and interleukin-2 (IL-2) but not IL-4 or IL-5. Isotype profiles of antibodies specific for VSG showed that IgG1, IgG2a, and IgG3 switch responses predominated; no VSG-specific IgE responses were detected. To determine whether cryptic IL-4 responses played a role in promoting the unexpected IgG1 switch response, IL-4 knockout mice were infected; the cytokine responses and Ig isotype profiles of IL-4 knockout mice were identical to those of the wild-type control mice except for dramatically reduced IgG1 levels in response to VSG. Thus, these results revealed an IL-4-dependent component of the VSG-driven B-cell Cmu-to-Cgamma1 switch. We speculate that an IL-4 response is mediated primarily by cells other than T lymphocytes since IL-4-secreting but parasite antigen-unresponsive, "background" cells were detected in all infected mice and since infected nude mice also displayed a detectable IgG1 switch response. Overall, our results suggest that B-cell clonal stimulation, maturation, and Ig class switching in African trypanosomiasis may be partially regulated by unusual mechanisms that do not include antigen-specific Th1 or Th2 cells.
No abstract available.
During the past decade, extensive knowledge has been gained with respect to the cellular and molecular mechanisms associated with variant surface glycoprotein (VSG) gene switching in trypanosomes. However, comparatively little is known about the cellular and molecular factors that regulate the host B-cell response to VSG determinants during infection. Here, John Mansfield reflects on the nature of this response.
Suppression of host T cell responses is one of the hallmarks of infection with the African trypanosomes. The cellular basis for immunosuppression includes the generation of suppressor macrophages that down-regulate T cell proliferative but not necessarily cytokine responses to both mitogen and trypanosome Ag. Since macrophages from infected animals display activation characteristics, we have asked whether products of activated cells, specifically nitric oxide (NO) and PG, may mediate the suppressor cell effects and immunosuppression observed. We demonstrate that cells isolated from B10.BR mice infected with Trypanosoma brucei rhodesiense exhibited transcriptional up-regulation of inducible NO synthase and released significant amounts of NO. The levels of NO released were elevated further after stimulation of cells with T cell mitogens or specific parasite Ag; antibody blocking experiments demonstrated that this up-regulation of NO synthesis was at least partially dependent upon IFN-gamma and TNF-alpha. The addition of inducible NO synthase substrate analogues such as NG-monomethyl-L-arginine to cell cultures inhibited NO release and also partially reversed the suppressor cell activity and immunosuppression displayed by such cultures. PG levels also were elevated in cell cultures from infected mice, but the PG inhibitor indomethacin had no effect on suppressor cells or suppression when added alone to the cultures. However, the concurrent inhibition of NO and PG synthesis by the addition of both NG-monomethyl-L-arginine and indomethacin completely blocked suppressor cell activity associated with infected macrophages and also resulted in further recovery of infected cells from immunosuppression, thus revealing an epistatic effect between these two mediators. We conclude that macrophage activation in trypanosomiasis induces the release of reactive nitrogen intermediates and PG, which down-regulate proliferative responses by T cells during infection.
No abstract available.
T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.
The characterization of B cell epitopes on the trypanosome variant surface glycoprotein (VSG) rests on elucidation of variant specific amino acid sequences that may be exposed or buried as a result of the natural conformation of these molecules in the surface coat. Despite the fact that different VSGs have heterogeneous primary sequences and unique antigenic characteristics, recent high resolution X-ray crystallographic analyses of VSGs have revealed a conserved 3-dimensional structure common to these surface proteins [19]. We took advantage of this conserved structural conformation to help predict which variant subregions of VSG molecules may contain exposed or buried variant specific B cell epitopes. Using Staden data tables, we aligned the deduced amino acid sequence of Trypanosoma brucei rhodesiense LouTat 1 VSG, a molecule that has been characterized immunologically in this laboratory, with 12 other complete VSG sequences including the T. b. brucei MiTat 1.2 VSG that has been characterized in crystallographic studies. Results of this analysis predict that there are eight defined clusters of variant amino acids which may contribute to exposed B cell epitopes, and ten defined clusters of variant amino acids which may contribute to buried B cell epitopes, on all VSG molecules. Interestingly, this analysis also revealed a VSG consensus sequence in which certain conserved motifs are present in all VSGs. The shared elements of VSG sequences corresponded to known secondary structures present in MiTat 1.2, and included groups of conserved amino acids responsible for turns in subregions of the protein, for structural positioning of the variable residues on the exposed surface, and for the dimerization of VSG monomers. Overall, these observations may aid in the targeting and mapping of exposed and buried VSG specific B cell epitopes, and also may offer clues as to elements of the primary sequence that are important for the conserved 3-dimensional structure of antigenically distinct VSG molecules.
The T-cell dependency of B-cell responses to variant surface glycoprotein (VSG) epitopes exposed in their native surface conformation on Trypanosoma brucei rhodesiense clone LouTat 1 was investigated. T-cell requirements were examined by analyses of gamma globulin preparations derived from trypanosome-infected BALB/c nude (nu/nu) and thymus-intact (nu/+) mice. A radioimmunoassay was used to selectively quantitate antibody binding to native VSG 1 epitopes present on the surface of viable trypanosomes. Such analyses of VSG-specific antibody in infected mice demonstrated that in the absence of T cells there was a significant B-cell response to exposed VSG epitopes; however, in the presence of T cells these surface epitope-specific responses were greatly enhanced. In contrast to infection, immunization of mice with purified VSG 1 or paraformaldehyde-fixed parasites elicited significant VSG surface epitope-specific responses only in the presence of T cells (i.e., in nu/+ mice only). VSG-specific antibody responses in mice infected with three other clonal T. brucei rhodesiense populations (LouTat 1.2, 1.5, and 1.9) were found to be similar in this pattern, although not identical, to the anti-LouTat 1 responses. An important exception was that mice infected with LouTat 1.8 required T cells to produce VSG surface-specific antibody. Thus, the VSG surface epitope-specific B-cell responses in trypanosome-infected mice represent composite T-cell-independent and T-cell-dependent processes, and a significantly stronger response is made in the presence of T cells. However, immunization with VSG in the absence of infection elicited only T-cell-dependent responses. Since the relative contribution of T-cell-independent and T-cell-dependent processes to the total VSG-specific antibody produced during infection was variable (as seen with the absence of a T-cell-independent response to LouTat 1.8), this may reflect differences in the primary structure or display of VSG molecules on the trypanosome membrane or may represent active parasite interference with some epitope-specific B-cell responses.
The current study examines the idiotypic expression and regulation of variant surface glycoprotein (VSG)-specific B cell responses during African trypanosomiasis. Utilizing competitive inhibition RIA analysis, we detected antibodies in the serum of BALB/cByJ mice infected with Trypanosoma brucei rhodesiense clone LouTat 1.5 that recognized the same VSG epitopes as three VSG 1.5-specific mAb. These epitope-specific antibody responses were detectable by day 5 of infection, peaked by day 10, and then declined slowly through day 15 of infection. VSG-specific antibodies detectable in the serum of infected BALB/cByJ mice included those that were idiotypically cross-reactive with the VSG 1.5-specific mAb. These idiotypically defined, VSG-specific antibody responses appeared to peak around day 7 of infection, but then declined to near preimmune levels by day 15 of infection, demonstrating that the aggregate epitope-specific response was composed only in part by the idiotypically cross-reactive responses. Although corresponding antiidiotypic antibodies could not be detected in infected sera during periods of up- or down-regulation of idiotypically defined antibodies, flow cytometry analysis of lymphocytes isolated from the spleens of LouTat 1.5-infected BALB/cByJ mice revealed the presence of antiidiotypic receptor-bearing cells. These cells were detectable primarily during days 10 to 12 of infection and subsequently down-regulated their receptors, or declined in numbers, to near preimmune levels by day 15 of infection. The appearance of these antiidiotypic receptor-bearing cells coincides with the decline of idiotypic antibody present in the serum of the LouTat 1.5-infected mice and may represent nascent evidence for idiotypic regulation of trypanosome-specific immune responses in infected animals.
Regulatory mechanisms governing B cell responses to the trypanosome variant surface glycoprotein (VSG) molecule currently are being studied. As a fundamental basis for examining such regulation, the epitope specificities and idiotypic profiles of murine mAb produced to the VSG of Trypanosoma brucei rhodesiense clone LouTat 1.5 were determined. Variant specific mAb were used to probe VSG proteolytic peptides in Western blot analysis, to serve as competitive inhibitors in RIA analyses with purified VSG molecules, and to examine membrane-binding patterns of labeled trypanosome cells in order to evaluate epitope specificities. By using these approaches, a conformational epitope expressed only on the VSG 1.5 surface coat of viable trypanosomes was detected, and two nonconformationally determined epitope clusters were recognized within the subsurface V region of the VSG 1.5 molecule. The subsurface epitope clusters may be repeated on the VSG molecule because each was present on more than one proteolytic VSG peptide fragment. Idiotypic profiles of selected VSG-specific mAb subsequently were determined with xenogeneic antiidiotypic typing sera. Results from competitive inhibition RIA analyses using these reagents demonstrated that varying levels of idiotypic cross-reactivity exist among the subsurface VSG epitope-specific mAb; this cross-reactivity extended to idiotope(s) expressed by a mAb recognizing a surface conformational epitope of the VSG 1.5 molecule. Analysis of complementary idiotypic/antiidiotypic antibody pairs revealed that these specific interactions were inhibited by purified VSG 1.5 but not by purified VSG 1.9, which was derived from a heterologous variant antigenic type. The model mAb described here, and reagents recognizing their idiotypic markers, comprise a foundation for analysis of idiotypic regulation of VSG-specific B cell responses during infection.
No abstract available.
Regulation of B cell responses to the trypanosome surface Ag was examined in H-2k compatible "responder" B10.BR and "nonresponder" C3H mice after infection with two variant clones of Trypanosoma brucei rhodesiense. Development of a selective RIA for independent detection of antibody binding to surface (exposed) and subsurface (buried) epitopes of the trypanosome variable surface glycoprotein (VSG) molecule permitted sensitive quantitation and kinetic characterization of immune responses to these epitopes. The infected B10.BR mice responded to both exposed and buried VSG epitopes of clone LouTat 1 trypanosomes, whereas a B cell response by C3H mice to exposed VSG epitopes was not detected by RIA analyses at any time. However, VSG-specific IgM and IgG responses were produced to buried VSG epitopes, demonstrating that LouTat 1 induced immunoregulation was specific only for the B cell responses to exposed VSG epitopes. Subsequently, comparisons of B10.BR and C3H B cell responses to a heterologous variant, LouTat 1.5, were made. The results revealed that both infected mouse strains produced VSG 1.5-specific antibody to exposed and buried epitopes with different kinetics and maximal sera concentrations, showing, therefore, that these responses are not coordinately regulated. In addition, it was clear that the observed immunosuppression to exposed LouTat 1 VSG epitopes in C3H mice could be regulated by the parasite since functional C3H B cell responses were mounted against exposed VSG epitopes of a closely related variant (LouTat 1.5) after infection.
The question of linkage of virulence traits to variable surface glycoprotein (VSG) expression in African trypanosomiasis was addressed. Previously we demonstrated that daughter cells arising in mice infected with a genetically homogeneous trypanosome population of Trypanosoma brucei rhodesiense were more virulent than the infecting population (J. A. Inverso and J. M. Mansfield, J. Immunol. 130:412, 1983). These virulent trypanosomes expressed differences in surface phenotype compared with the infecting variant types, and we proposed that virulence may be "linked" to VSG expression. In the present study, however, we have shown that expression of virulence is independent of the VSG phenotype displayed by trypanosome populations. A VSG-identical but highly virulent subpopulation of T. b. rhodesiense LouTat 1 was derived by rapid subpassage and subcloning in immunosuppressed mice. The virulent LouTat 1A subclone derived in this manner killed B10.BR/SgSnJ mice in 3 to 4 days postinfection compared with approximately 60 days for the parent clone, LouTat 1. The virulent subclone LouTat 1A appears to express the same VSG as the less virulent LouTat 1 population, as determined by polyspecific and monoclonal antibody-binding assays, cross-protection tests, and amino acid sequence analyses of the N-terminal portion of the VSG molecules. When LouTat 1 and subclone LouTat 1A were injected into a heterologous host species, multiple variant antigenic types (VATs) arising from each inoculum were isolated and characterized. VATs derived from the virulent subclone were as uniformly virulent for B10.BR mice as LouTat 1A. In summary, these results demonstrate that trypanosome virulence, once expressed, is a stable phenotype that does not seem to be associated with a particular VSG phenotype, nor does virulence change with the expression of different VSG genes.
The question of genetic linkage of parasite-specific immune responses to resistance to infection in experimental African trypanosomiasis was addressed. For this purpose, major histocompatibility complex-compatible resistant and susceptible inbred mouse strains and their F1 hybrid, F2 hybrid, and backcross offspring were infected with Trypanosoma brucei rhodesiense LouTat 1. Immunologic control of the first peak of parasitemia and survival times were the parameters measured. As we have reported previously (R. F. Levine and J. M. Mansfield, J. Immunol. 133:1564, 1984), B10.BR/SgSnJ mice are relatively resistant and controlled the growth of the infecting variant antigenic type (VAT) by mounting an antibody response to exposed epitopes of the variable surface glycoprotein (VSG). Fluctuating parasitemias resulting from sequential growth of different variable antigenic types occurred subsequently, and these mice died with a median survival time of 48 days. C3HeB/FeJ mice, relatively susceptible, did not control the infecting VAT and did not exhibit VSG-specific antibodies. These mice died with a median survival time of 22 days. The (B10.BR X C3H)F1 hybrids derived from crosses between resistant and susceptible mice all exhibited VSG-specific antibody responses and controlled the infecting VAT population. However, the median survival time of the F1 hybrids (24 days) was not significantly different from the survival time of the susceptible C3H parent. These findings demonstrate for the first time that antibody-mediated control of parasitemia is inherited as a dominant trait; that overall resistance, as measured by survival time, is inherited as a recessive trait (e.g., susceptibility is dominant); and that the two events segregate independently of one another. Further analyses of the inheritance of immunity and resistance (survival time) were made in which the F2 hybrid and backcross studies revealed that there are multiple genes controlling the VSG-specific antibody response as well as determining susceptibility. An extension of the present studies to a similar but non-major histocompatibility complex-mouse model system of resistance and susceptibility (C57BL/6J and C3H/HeJ mice, F1 hybrids, and 11 recombinant inbred B X H strains derived from them) was made in order to link the strain distribution patterns of known genetic markers with control of VSG-specific antibody responses or with control of susceptibility. Results of this study showed that resistance varied independently of the ability to control parasitemia with VSG-specific B cell responses.(ABSTRACT TRUNCATED AT 400 WORDS)
