Allogeneic Ligand Research Articles

Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.

Generation of human alloantigen-specific T cells from peripheral blood.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

The Ability To Rearrange Dual TCRs Enhances Positive Selection, Leading to Increased Allo- and Autoreactive T Cell Repertoires

Thymic selection is designed to ensure TCR reactivity to foreign Ags presented by self-MHC while minimizing reactivity to self-Ags. We hypothesized that the repertoire of T cells with unwanted specificities such as alloreactivity or autoreactivity are a consequence of simultaneous rearrangement of both TCRα loci. We hypothesized that this process helps maximize production of thymocytes capable of successfully completing thymic selection, but results in secondary TCRs that escape stringent selection. In T cells expressing two TCRs, one TCR can mediate positive selection and mask secondary TCR from negative selection. Examination of mice heterozygous for TRAC (TCRα(+/-)), capable of only one functional TCRα rearrangement, demonstrated a defect in generating mature T cells attributable to decreased positive selection. Elimination of secondary TCRs did not broadly alter the peripheral T cell compartment, though deep sequencing of TCRα repertoires of dual TCR T cells and TCRα(+/-) T cells demonstrated unique TCRs in the presence of secondary rearrangements. The functional impact of secondary TCRs on the naive peripheral repertoire was evidenced by reduced frequencies of T cells responding to autoantigen and alloantigen peptide-MHC tetramers in TCRα(+/-) mice. T cell populations with secondary TCRs had significantly increased ability to respond to altered peptide ligands related to their allogeneic ligand as compared with TCRα(+/-) cells, suggesting increased breadth in peptide recognition may be a mechanism for their reactivity. Our results imply that the role of secondary TCRs in forming the T cell repertoire is perhaps more significant than what has been assumed.

Solution mapping of T cell receptor docking footprints on peptide-MHC.

T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a "recognition code" between conserved elements of the TCR and MHC genes. Although approximately 17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2Ld-QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.

Alloreactivity between Disparate Cognate and Allogeneic pMHC-I Complexes Is the Result of Highly Focused, Peptide-dependent Structural Mimicry

Our understanding of the molecular mechanisms of T cell alloreactivity remains limited by the lack of systems for which both the T cell receptor allo- and cognate ligand are known. Here we provide evidence that a single alloreactive T cell receptor interacts with analogous structural regions of its cognate ligand, HLA-B*0801(FLRGRAYGL), as its allogeneic ligand, HLA-B*3501(KPIVVLHGY). The crystal structures of the binary peptide-major histocompatibility complexes show marked differences in the conformation of the heavy chains as well as the bound peptides. Nevertheless, both epitopes possess a prominent solvent-exposed aromatic residue at position 7 flanked by a small glycine at position 8 of the peptide determinant. Moreover, regions of close structural homology between the heavy chains of HLA B8 and HLA B35 coincided with regions that have previously been implicated in "hot spots" of T cell receptor recognition. The avidity of this human T cell receptor was also comparable for the allo- and cognate ligand, consistent with the modes of T cell receptor binding being broadly similar for these complexes. Collectively, it appears that highly focused structural mimicry against a diverse structural background provides a basis for the observed alloreactivity in this system. This cross-reactivity underpins the T cell degeneracy inherent in the limited mature T cell repertoire that must respond to a vast diversity of microbial antigens.

Allogeneic and syngeneic class I MHC complexes drive the association of CD8 and TCR on 2C T cells

In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8 + T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L d exhibits a less significant role for CD8 than recognition of the syngeneic MHC K b. Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.

Potent cytolytic response by a CD8+ CTL clone to multiple peptides from the same protein in association with an allogeneic class I MHC molecule.

CTL clone 2C recognizes the allogeneic class I MHC molecule L(d) in association with peptides derived from alpha-ketoglutarate dehydrogenase (oxoglutarate dehydrogenase (OGDH)), a ubiquitous intracellular protein. One of these peptides, QLSPFPFDL (QL9), elicits more vigorous cytolytic responses than two previously identified naturally processed peptides with overlapping sequences, LSPFPFDL (p2Ca) and VAITRIEQLSPFPFDL (p2Cb), from OGDH. In this study, we show that QL9 forms a more stable complex with cell surface L(d) than does p2Ca or p2Cb and is processed from the longer, naturally occurring peptide p2Cb by 20S proteosomes in vitro. The N-terminal cyclized pyroglutaminyl QL9 (pyroQL9), a form of QL9 to which it is converted at the low pH used for peptide isolation from tissue extracts, is even more active than QL9 in cytotoxicity assays with 2C CTL. Overall, the results indicate that along with the abundant natural peptides p2Ca and p2Cb, the QL9 and other OGDH peptides of various lengths, sharing a conserved C-terminal sequence, are also processed and presented with L(d) as allogeneic ligands for T cells expressing 2C TCR. All these peptides, each available in a low amount, could act in concert at the cell surface, resulting in a high density of cognate ligands that accounts for the exceptionally potent cytolytic response by 2C CTL.

Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization.

Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Cytotoxic T lymphocytes show HLA-C-restricted recognition of EBV-bearing cells and allorecognition of HLA class I molecules presenting self-peptides.

Human CTL have been isolated that show self-restricted recognition of autologous lymphoblastoid cell lines and allorecognition. The lymphoblastoid cell line ligand most likely used a peptide that is expressed in EBV-bearing cells when the virus enters the lytic cycle. This peptide is presented to CD8+ CTL by HLA-Cw7 molecules. The allogeneic ligand recognized on non-EBV-infected cells is composed of a class I glycoprotein and a naturally selected self-peptide. In previous studies we demonstrated that this ligand is determined by two MHC-linked genes: one gene encodes the allogeneic class I molecule whereas the other controls the self-peptide. Despite the use of different peptides and different class I molecules, seemingly equivalent structures are formed that enable these two ligands to function as antigenic mimics of each other. CTL with the same patterns of dual specificity could be isolated from four unrelated donors, indicating that HLA-Cw7 is frequently involved in self-restricted recognition of EBV-harboring cells. Such CTL could help not only to contain lytic virus during a primary infection but also may be maintained life-long to eliminate cells in which reactivated virus appears.

T cells can distinguish between allogeneic major histocompatibility complex products on different cell types

In the response of T cells to foreign antigens, the ligand for the T cell alpha/beta receptor is presented on a cell surface as a fragment of antigen complexed to one of the membrane molecules encoded in the major histocompatibility complex (MHC). The receptor apparently interacts via its variable elements (V beta, D beta, J beta, V alpha and J alpha) with residues within both the antigen and MHC portion of the ligand. The frequency of T cells responding to a conventional antigen plus self MHC is usually quite low, presumably reflecting the relative rarity of receptors with the particular combination of variable elements to match the antigen/MHC ligand. T cells also respond to allogeneic forms of MHC molecules in the absence of added antigen. In this case the frequency of responding T cells is very high. One hypothesis to explain this observation is that, in the absence of foreign antigen, MHC molecules are complexed to a large array of peptides derived from self-proteins. In this case the combination of the polymorphic MHC amino acid residues and many different self peptides presents so many possible ligands that the likelihood of recognition by a given T cell receptor is quite high. The recent crystallography experiments which revealed a dramatic binding cleft on the face of a human MHC molecule have given impetus to this view, but as yet there is no direct supporting evidence. We have recently described a close association between murine T cell receptors utilizing the V beta 17a element and reactivity to various allogeneic forms of the murine MHC molecule, I-E (ref. 8). In this paper, we show that this I-E ligand is detected on B cells, but not on I-E+ macrophages or fibroblasts expressing a transfected I-E gene. These results strongly suggest a B cell specific product combines with I-E to form the allogeneic ligand for V beta 17a+ receptors and thus support the concept of alloreactivity described above.