Analyses of VSG sequence and structure have influenced current thinking and experimental approaches to understanding B- and T-cell responses to this protective antigen during infection with African trypanosomes. Comparative crystallographic analyses and molecular modeling of Trypanosoma brucei
VSG molecules revealed that antigenically distinct VSGs may fold into very similar structures (3
). This structural conservation may reflect a need to maintain dense packing of VSGs within the trypanosome surface coat during switching, when new VSG homodimers intercalate into the existing coat structure. Furthermore, maintenance of a dense and rigid molecular surface coat may be important both for limiting B-cell responses to surface solvent-exposed epitopes and for providing a T-cell-independent signal to B cells during early trypanosome population outgrowth (18
Another level of “conservation,” namely, the conservation of HV regions at specific sites within the general structure of related molecules, has been noted among different VSGs. Identification of two major HV regions (HV-2 and -3) that are solvent exposed suggests that selection for amino acid hypervariability within such regions in different VSGs may have occurred in order to evade antibody-mediated destruction (19
). Another HV region, HV-1, located within an amphipathic α-helix of VSG monomers that is not exposed on the VSG surface coat (Fig. ), is also conserved among structurally related VSGs and is predicted to have been selected by Th-cell pressure (3
). The conservation of both VSG structure and sites of amino acid hypervariability within a structural motif that displays predicted and functional peptide binding to MHC-II molecules led to the hypothesis that different VSGs may be processed similarly so as to preferentially present peptides from the HV-1 region to Th cells. Thus, our studies of Th-cell specificity were designed to determine whether functional and protective MHC-II-restricted Th-cell responses to the VSG molecule are generated primarily against HV-1 region epitopes.
Th-cell lines and hybridomas were derived from VSG-specific Th cells activated during infection of relatively resistant B10.BR mice in order to map the specificity of these cells for discreet peptide sequences within the LouTat 1 VSG molecule. Our initial experiments using VSG-specific Th-cell lines suggested that Th cells may respond to peptide fragments that map outside the HV-1 subregion. Further analysis using VSG-specific T-cell hybridomas and overlapping peptides spanning the LouTat 1 VSG sequence demonstrated specificity for multiple sites that were present throughout the N-terminal domain of the molecule. The number of different TCR Vβ chains expressed by VSG-specific T-cell hybridomas and the different patterns of peptide reactivity expressed by those T-cell hybridomas displaying identical Vβ chains (e.g., presumptive evidence for distinct Vα chains) clearly demonstrate that the VSG-specific T-cell response is not mono- or oligoclonal; such limited clonality might have been expected if T-cell responses were limited to a distinct HV subregion of VSG.
Confirmation of the T-cell hybridoma results was obtained by directly determining the VSG peptide-specific responses of infected mouse T cells, which showed that the same subsets of peptides were recognized by Th1 cells activated in situ and also that additional minor Th1-cell-reactive sites were detectable. Several T-cell responses mapped to peptides that overlapped with the HV-1 subregion, but the majority of T-cell-reactive sites were distributed throughout the N-terminal domain of the molecule, including the HV-2 and HV-3 subregions (Fig. ).
It is unclear from these experiments whether all potential epitopes within the HV-1 region are processed or presented to T cells during infection. The specificity of several T-cell hybridomas, including two that recognize VSG peptides expressed at high levels in vivo (11
), could not be identified using the overlapping VSG peptides prepared for this study. Additionally, some peptides may be expressed at levels too low to detect with the hybridomas in vitro or may be recognized by Th cells that did not withstand the hybridoma selection process. Therefore, the evolutionary advantage provided by maintenance of the HV-1 region has yet to be determined. As Field and Boothroyd (19
) proposed, the amphipathic α-helix containing the HV-1 subregion may simply be able to accept extensive amino acid variation without detriment to the overall structure, or this region may also be involved in B-cell-specific responses, despite the fact that it is predicted to be buried in the surface coat structure. It is possible that early T-cell-independent B-cell responses to solvent-exposed HV-2 and HV-3 epitopes of the surface coat (18
) cause molecular “tufting” of the structure, which exposes regions of the VSG normally buried within the coat. One of these regions may be the HV-1 region.
Since some T-cell responses to VSG epitopes map outside the putative HV regions, one might predict a level of VSG cross-reactivity among antigenically distinct VSGs. Functionally, however, there is no evidence for such cross-reactivity in these experiments (see Fig. , in which there is no demonstrable cross-reactivity of LouTat 1 VSG-specific T cells with the closely related T. b. brucei
117 VSG molecule) (18
) or in our earlier VSG-specific Th-cell studies (40
). Thus, T-cell-reactive sites in VSGs probably represent variant sequence microheterogeneity among antigenically distinct VSGs (38
). Aggregate VSG sequence database entries, as well as recent surveys of VSGs expressed by many different field isolates that included closely related trypanosome strains, suggest that such microheterogeneity is widespread in the N-terminal domains of VSGs (21
). The data presented in our current study provide the functional evidence for such heterogeneity in terms of T-cell recognition.
Thus, it appears that natural selection for microheterogeneity throughout the VSG molecule resulted from B- and T-cell responses to exposed and buried residues. Interestingly, these findings bring into question the biological relevance of VSG gene modification resulting from segmental conversion among different silent or basic-copy VSG genes (2
). New VSG genes resulting from such segmental conversion would contain large segments of distinct VSGs that, if expressed, would have to be unique in primary sequence in order to evade B- and T-cell recognition. The only way that such variant sequences could be expressed productively on viable trypanosomes during an ongoing infection would be if T cells were limited to recognizing specific (e.g., HV) subregions; otherwise, all potential target peptides in the new and old segments would have to differ for each such variant that arises. Based on our current results, therefore, we suggest that such segmental conversion among VSG genes would likely have a nonviable outcome for trypanosomes that express them during long-term infection in immunocompetent hosts.
The observation that VSG-specific T-cell responses are temporally limited during infection, being detectable within a few days of infection but largely undetectable after 10 days, has several bases. First, parasitemia representing the infecting LouTat 1 trypanosome population was eliminated by day 6 postinfection. Any parasites appearing after this time (e.g., on or after day 10) would be antigenic variants displaying a new VSG surface coat. Thus, VSG peptides derived from LouTat 1 would no longer be expressed significantly on APCs ex vivo. This has been explored in detail in recent studies in our laboratory (11
; Freeman et al., unpublished data). We also have shown that exposure of APCs to glycosylphosphatidylinositol residues of shed VSG renders the cells gradually unable to produce stable intracellular MHC-II-peptide complexes (Freeman et al., unpublished data). Thus, the absence of the prior VSG coupled with alterations in APC function contributes to the temporal nature of VSG-specific T-cell responses during infection.
Th-cell epitope selection may be influenced by a number of factors leading to TCR recognition of peptide-MHC-II complexes, including the antigen processing events that produce antigenic fragments and the binding of specific peptides within the MHC-II groove. Structural features such as disulfide bonds and noncovalent interactions influencing antigen stability have been associated with the efficacy of antigen presentation and epitope selection (7
). In addition, immunodominant T-cell epitopes have been associated with structurally stable sites in protein antigens; these sites are often flanked by unstable regions predicted to be preferentially cleaved during processing, thereby allowing adjacent stable regions to be exposed for binding to MHC-II (12
). The stable sites could exist as a range of structures but tended to be hydrophobic due to the importance of hydrophobic residues in peptide binding to MHC-II (24
). Although hydrophobic interactions may bias Th-cell epitopes to the protein interior, it has been suggested that peripheral epitopes may also bind MHC-II prior to proteolytic events that result in protein unfolding; the early binding to MHC-II could then prevent degradation of these epitopes during antigen processing in endosomal and lysosomal compartments (16
). Thus, theoretically, as shown by predicted epitope and MHC-II binding motifs, and practically, as shown by functional T-cell epitope mapping in this study, a variety of peptides from different non-HV-1-specific subregions of VSG are presented to T cells (Fig. ).
The absence of T-cell specificity for the relatively invariant C-terminal domain of VSGs is another key finding of this study. The fact that T cells did not recognize peptides from this domain in the peptide mapping approach is confirmed by the absence of any detectable cross-reactivity at the functional level when examining T-cell responses to different VSGs that share C-terminal domain homology (40
) (Fig. ). In retrospect, it may be that an absence of T-cell reactivity to the C-terminal domain, coupled with the natural inaccessibility of antibodies to this buried region, resulted in a lack of selection for microheterogeneity within the C-terminal domain, thus largely maintaining sequence homogeneity within VSG type and class. The mechanism(s) that prevents potential T-cell recognition of this domain is unknown at present, but our recent studies on modulation of APC function during trypanosomiasis show a progressive inability to process and present new VSG peptides (11
; Freeman et al., unpublished data). There is also the possibility that the C-terminal domain of VSGs may not be accessible to the enzymes that degrade the rest of the molecule during uptake and processing. Ultimately, these types of events may predispose APCs to display only a subset of N-terminal domain VSG peptides during infection. Thus, our work at once experimentally refutes the HV-1 subregion hypothesis but reveals, ironically, that the relatively invariant C-terminal subregion is not recognized by T cells. In other words, VSG-specific T-cell responses are limited to certain subregions, just not the one defined by the HV-1 hypothesis.
Overall, and in summary, this is the first study to characterize the specificity of Th cells activated during infection for distinct peptides of the VSG molecule. The demonstration that peptides from the HV-1 (or other HV) subregion are not preferentially presented to T cells during infection is a finding that prompts rethinking of the evolutionary advantage that this buried HV region may provide to the parasite, as well as the overall implications for conserved structure on processing and presentation of VSG peptides. Further analysis of Th-cell specificity and the identities of VSG-derived peptides presented during ongoing infection is necessary to understand how VSG is processed and presented by APCs, to identify the spectrum of immunodominant epitopes in different VSGs, and to understand the significance, if any, of the HV-1 region for host immune resistance. The discovery that T cells fail to recognize peptides associated with the relatively conserved C-terminal domain of VSGs opens the door to experimental approaches that may preferentially stimulate Th cells to provide cross-variant protection to the infected host. This is theoretically possible because although antibodies cannot access buried residues of the C-terminal domain, T cells could be generated specifically against peptides derived from this domain to provide variant cross-protection during African trypanosomiasis.