Human Vγ2Vδ2+ T cells proliferate in vivo during many microbial infections. We have found that Vγ2Vδ2+ T cells recognize nonpeptide prenyl pyrophosphates and alkylamines. We now have defined structural features that determine the antigenicity of prenyl pyrophosphates by testing synthetic analogs for bioactivity. We find that the carbon chain closest to the pyrophosphate moiety plays the major role in determining bioactivity. Changes in this area, such as the loss of a double bond, abrogated bioactivity. The loss of a phosphate from the pyrophosphate moiety also decreased antigenicity 100- to 200-fold. However, nucleotide monophosphates could be added with minimal changes in bioactivity. Longer prenyl pyrophosphates also retained bioactivity. Despite differences in CDR3 sequence, Vγ2Vδ2+ clones and a transfectant responded similarly. Ag docking into a Vγ2Vδ2 TCR model reveals a potential binding site in germline regions of the Vγ2Jγ1.2 CDR3 and Vδ2 CDR2 loops. Thus, Vγ2Vδ2+ T cells recognize a core carbon chain and pyrophosphate moiety. This recognition is relatively unaffected by additions at distal positions to the core Ag unit.

We have previously found that monkey Vγ2Vδ2+ T cells mount adaptive immune responses in response to Mycobacterium bovis bacillus Calmette-Guérin infections. We have now analyzed rhesus monkey γδ T cell responses to nonpeptide Ags and superantigens. Like human Vγ2Vδ2+ T cells, rhesus monkey γδ T cells are stimulated when exposed to prenyl pyrophosphate, bisphosphonate, and alkylamine Ags. Responsiveness was limited to γδ T cells expressing Vγ2Vδ2 TCRs. Rhesus monkey Vγ2Vδ2+ T cells also responded to the superantigen, staphyloccocal enterotoxin A. Sequencing of the rhesus monkey Vγ2Vδ2 TCR revealed a strong sequence homology to human Vγ2Vδ2 TCR that preserves important sequence motifs. Moreover, chimeric TCRs that pair human Vγ2 with monkey Vδ2 and monkey Vγ2 with human Vδ2 retain reactivity to nonpeptide Ags and B cell lymphomas. A molecular model of the rhesus monkey Vγ2Vδ2 TCR has a basic region in the complementarity-determining region 3 binding groove that is similar to that seen in the human Vγ2Vδ2 TCR and preserves the topology of the complementarity-determining region loops. Thus, recognition of nonpeptide prenyl pyrophosphate, bisphosphonate, and alkylamine Ags is conserved in primates suggesting that primates can provide an animal model for human γδ T cell Ag responses.

Vγ2Vδ2 T cells comprise the major subset of peripheral blood γδ T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the Vγ2Vδ2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the Vγ2Vδ2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C5-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to γδ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β2-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C5-OPP. Finally, pulsing of BZ-(C)-C5-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.

SummaryT cells bearing y/b antigen receptors comprise a resident population of intraepithelial lymphocytes in organs such as skin, gut, and lungs, where they are strategically located to contribute to the initial defense against infection . An important unsolved question about antigen-driven y/b T cell responses regards the breadth of their T cell receptor (TCR) repertoire, since many specific epithelial compartments in mice display limited diversity. We have examined the diversity of TCR S gene expression among human y/b T cells from skin lesions induced by intradermal challenge with Mycobacterium leprae . We show that the vast majority of'y/b cells from M. leprae lesions use either V61-J61 or V62 -J61 gene rearrangements and, within a given region of the lesion, display limited junctional diversity. This contrasts markedly with the extensive diversity of y/b T cells from peripheral blood of these same individuals, as well as skin from normal donors . These results indicate that they/b response to M. leprae involves the selection of a limited number of clones from among a diverse repertoire, probably in response to specific mycobacterial and/or host antigens . O ne of the fundamental aspects of cellular immunology is the selection and clonal expression of T cells bearing specific receptors by antigens. While the selection of ci/(3 T cells has been studied in detail, little is known about the selection of y/b T cells by antigen . The genetic diversity of the TCR provides a measure of the scope of the T cell repertoire. In contrast to the a/a TCR, the germline gene segment diversity for both the TCR y and S chains is small . The further limitation of this diversity by the preferential usage of only a few variable (V) genes or V gene pairs at specific anatomical locations (1-3) suggests a particularly narrow TCR repertoire, likely reflecting recognition of a limited number of ligands . On the other hand, there is unprecedented junctional diversity, particular in the b chains of this receptor (4-6) . We reasoned that analysis of the TCR y/b repertoire of a specific immune response would provide clues about the set of antigens recognized : whether diverse or limited, conventional, or superantigen-like. The presence of antigen-reactive TCR -y/6-bearing cells in leprosy skin lesions (7) provides a unique in vivo model to examine the y/b receptor repertoire at the site of immunopathologic reaction .Much experimental evidence indicates that y/b T cells contribute to the granuloma formation in response to mycobacterial infection (7) . First, TCR y/b cells comprise a strikingly high percentage of the T cell population in infectious disease lesions that contain recently formed granulomas. These include lepromin skin tests (Mitsuda reactions), which are experimental DTH reactions induced by intradermal injection of Mycobacterium lepme, reversal reactions in leprosy, which represent a naturally occurring DTH response to M. leprae, and localized American cutaneous leishmaniasis. Second, the y/b T cells from these infectious l...