Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.

T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.

T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex.

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

Identification of multiple public TCR repertoires in chronic beryllium disease.

Chronic beryllium disease (CBD) is a granulomatous lung disease characterized by the accumulation of beryllium (Be)-specific CD4(+) T cells in bronchoalveolar lavage. These expanded CD4(+) T cells are composed of oligoclonal T cell subsets, suggesting their recruitment to the lung in response to conventional Ag. In the current study, we noted that all bronchoalveolar lavage-derived T cell lines from HLA-DP2-expressing CBD patients contained an expansion of Be-responsive Vß5.1(+) CD4(+) T cells. Using Be-loaded HLA-DP2-peptide tetramers, the majority of tetramer-binding T cells also expressed Vß5.1 with a highly conserved CDR3ß motif. Interestingly, Be-specific, Vß5.1-expressing CD4(+) T cells displayed differential HLA-DP2-peptide tetramer staining intensity, and sequence analysis of the distinct tetramer-binding subsets showed that the two populations differed by a single conserved amino acid in the CDR3ß motif. TCR Vα-chain analysis of purified Vß5.1(+) CD4(+) T cells based on differential tetramer-binding intensity showed differing TCR Vα-chain pairing requirements, with the high-affinity population having promiscuous Vα-chain pairing and the low-affinity subset requiring restricted Vα-chain usage. Importantly, disease severity, as measured by loss of lung function, was inversely correlated with the frequency of tetramer-binding CD4(+) T cells in the lung. Our findings suggest the presence of a dominant Be-specific, Vß5.1-expressing public T cell repertoire in the lungs of HLA-DP2-expressing CBD patients using promiscuous Vα-chain pairing to recognize an identical HLA-DP2-peptide/Be complex. Importantly, the inverse relationship between expansion of CD4(+) T cells expressing these public TCRs and disease severity suggests a pathogenic role for these T cells in CBD.

Induced sensitization of tumor stroma leads to eradication of established cancer by T cells.

Targeting cancer cells, as well as the nonmalignant stromal cells cross-presenting the tumor antigen (Ag), can lead to the complete destruction of well-established solid tumors by adoptively transferred Ag-specific cytotoxic T lymphocytes (CTLs). If, however, cancer cells express only low levels of the Ag, then stromal cells are not destroyed, and the tumor escapes as Ag loss variants. We show that treating well-established tumors expressing low levels of Ag with local irradiation or a chemotherapeutic drug causes sufficient release of Ag to sensitize stromal cells for destruction by CTLs. This was shown directly using high affinity T cell receptor tetramers for visualizing the transient appearance of tumor-specific peptide-MHC complexes on stromal cells. Maximum loading of tumor stroma with cancer Ag occurred 2 d after treatment and coincided with the optimal time for T cell transfer. Under these conditions, tumor rejection was complete. These findings may set the stage for developing rational clinical protocols for combining irradiation or chemotherapy with CTL therapy.

Mutagenesis of beryllium-specific TCRs suggests an unusual binding topology for antigen recognition.

Unconventional Ags, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of Be-specific TCRs derived from the lung of a chronic beryllium disease patient. These TCRs are HLA-DP2-restricted and express nearly identical TCR Vß5.1 chains coupled with different TCR α-chains. Site-specific mutagenesis of all amino acids comprising the CDRs of the TCRA and TCRB genes showed a dominant role for Vß5.1 residues in Be recognition, with little contribution from the TCR α-chain. Solvent-exposed residues along the α-helices of the HLA-DP2 α- and ß-chains were also mutated to alanine. Two ß-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition with no contribution from the HLA-DP2 α-chain. These findings suggest that Be-specific T cells recognize Ag using an unconventional binding topology, with the majority of interactions contributed by TCR Vß5.1 residues and the HLA-DP2 ß1-chain. Thus, unusual docking topologies are not exclusively used by autoreactive T cells, but also for the recognition of unconventional metal Ags, such as Be.

Cutting edge: inhibitory effects of CD4 and CD8 on T cell activation induced by high-affinity noncognate ligands.

It has been proposed that MHC restriction during thymocyte selection is controlled by coreceptor (CD4 or CD8) sequestration of the signaling molecule Lck. We explored this model as a mechanism for preventing peripheral T cell activation due to non-MHC ligand cross-reactivities of TCRs. TCRs that have a range of affinities for a class I MHC ligand were transduced into a T cell hybridoma in the absence or presence of coreceptors. High and intermediate affinity TCRs (K(D) = 17 and 540 nM) did not require CD8 for T cell activity, but CD4 acted as a potent inhibitor of the intermediate affinity TCR. These and other findings support the view that even high-affinity TCR:ligand interactions can be influenced by coreceptor sequestration of Lck. Thus, CD4 and CD8 act as "coreceptor inhibitors" to maintain appropriate TCR-mediated MHC restriction in peripheral T cell activity.

Recurrence of intracranial tumors following adoptive T cell therapy can be prevented by direct and indirect killing aided by high levels of tumor antigen cross-presented on stromal cells.

Elimination of peripheral tumors by adoptively transferred tumor-specific T cells may require killing of cancer cells and tumor stromal cells. Tumor Ags are cross-presented on stromal cells, resulting in direct cytotoxic T cell (CTL) killing of both Ag-expressing cancer cells and stromal cells. Indirect killing of Ag loss variant cells also occurs. We show here that similar processes occur in a brain tumor stromal environment. We used murine cancer cell lines that express high or low levels of a peptide Ag, SIYRYYGL (SIY), recognized by transgenic 2C CD8(+) T cells. The two cell lines are killed with equivalent efficiency by 2C T cells in vitro. Following adoptive transfer of 2C T cells into mice with established SIY-Hi or SIY-Lo brain tumors, tumors of both types regressed, but low-Ag-expressing tumors recurred. High-Ag-expressing tumors contained CD11b(+) cells cross-presenting SIY peptide and were completely eliminated by 2C T cells. To further test the role of cross-presentation, RAG1(-/-) H-2(b) mice were infused with H-2(k) tumor cells expressing high levels of SIY peptide. Adoptively transferred 2C T cells are able to kill cross-presenting H-2(b) stromal cells but not H-2(k) tumor cells. In peripheral models, this paradigm led to a small static tumor. In the brain, activated 2C T cells were able to kill cross-presenting CD11b(+) cells and completely eliminate the H-2(k) tumors in most mice. Targeting brain tumor stroma or increasing Ag shedding from tumor cells to enhance cross-presentation may improve the clinical success of T cell adoptive therapies.

Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Different strategies adopted by K(b) and L(d) to generate T cell specificity directed against their respective bound peptides.

Mouse T cell clone 2C recognizes two different major histocompatibility (MHC) ligands, the self MHC K(b) and the allogeneic MHC L(d). Two distinct peptides, SIY (SIYRYYGL) and QL9 (QLSPFPFDL), act as strong and specific agonists when bound to K(b) and L(d), respectively. To explore further the mechanisms involved in peptide potency and specificity, here we examined a collection of single amino acid peptide variants of SIY and QL9 for 1) T cell activity, 2) binding to their respective MHC, and 3) binding to the 2C T cell receptor (TCR) and high affinity TCR mutants. Characterization of SIY binding to MHC K(b) revealed significant effects of three SIY residues that were clearly embedded within the K(b) molecule. In contrast, QL9 binding to MHC L(d) was influenced by the majority of peptide side chains, distributed across the entire length of the peptide. Binding of the SIY-K(b) complex to the TCR involved three SIY residues that were pointed toward the TCR, whereas again the majority of QL9 residues influenced binding of TCRs, and thus the QL9 residues had impacts on both L(d) and TCR binding. In general, the magnitude of T cell activity mediated by a peptide variant was influenced more by peptide binding to MHC than by binding the TCR, especially for higher affinity TCRs. Findings with both systems, but QL9-L(d) in particular, suggest that many single-residue substitutions, introduced into peptides to improve their binding to MHC and thus their vaccine potential, could impair T cell reactivity due to their dual impact on TCR binding.

T cell recognition of beryllium.

Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by a hypersensitivity to beryllium and characterized by the accumulation of beryllium-specific CD4(+) T cells in the lung. Genetic susceptibility to beryllium-induced disease is strongly associated with HLA-DP alleles possessing a glutamic acid at the 69th position of the ß-chain (ßGlu69). The structure of HLA-DP2, the most prevalent ßGlu69-containing molecule, revealed a unique solvent-exposed acidic pocket that includes ßGlu69 and represents the putative beryllium-binding site. The delineation of mimotopes and endogenous self-peptides that complete the αßTCR ligand for beryllium-specific CD4(+) T cells suggests a unique role of these peptides in metal ion coordination and the generation of altered self-peptides, blurring the distinction between hypersensitivity and autoimmunity.

Linking genetic susceptibility and T cell activation in beryllium-induced disease.

Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium (Be) exposure in the workplace. It is characterized by the accumulation of Be-specific CD4(+) T cells in the lung as well as persistent lung inflammation, culminating in the development of lung fibrosis. CBD occurs in 2 to 16% of Be-exposed workers depending on the individuals' genetic susceptibility and the characteristics of the exposure. Genetic susceptibility to Be-induced disease has been linked to major histocompatibility complex class II molecules. In particular, HLA-DP alleles possessing a glutamic acid at the 69th position of the beta-chain (betaGlu69) are most strongly linked to disease susceptibility. The HLA-DP alleles that present Be to T cells match those implicated in the genetic susceptibility, suggesting that the HLA contribution to disease is based on the ability of those molecules to bind and present Be to T cells. However, the structural features of betaGlu69-containing HLA-DP molecules that explain the disease association remain unknown. We have recently crystallized HLA-DP2, which is the most prevalent of the betaGlu69-containing HLA-DP molecules. Its unique structure, which includes surface exposure of betaGlu69, provides an explanation of the genetic linkage between betaGlu69-containing HLA-DP alleles and Be-induced disease.

Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity.

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9-L(d), we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep-L(d) complex (e.g. cell surface t(1/2)-values of 13 min for QL9-L(d) versus 87 min for F5R-L(d)). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R-L(d) compared to QL9-L(d) (K(D)=300 microM and K(D)=1.6 microM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R-L(d) (K(D)=1.2 and 6.3 microM). T cells that expressed these higher affinity TCRs were stimulated by F5R-L(d) in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.

Equilibrium between host and cancer caused by effector T cells killing tumor stroma.

The growth of solid tumors depends on tumor stroma. A single adoptive transfer of CD8(+) CTLs that recognize tumor antigen-loaded stromal cells, but not the cancer cells because of MHC restriction, caused long-term inhibition of tumor growth. T cells persisted and continuously destroyed CD11b(+) myeloid-derived, F4/80(+) or Gr1(+) stromal cells during homeostasis between host and cancer. Using high-affinity T-cell receptor tetramers, we found that both subpopulations of stromal cells captured tumor antigen from surrounding cancer cells. Epitopes on the captured antigen made these cells targets for antigen-specific T cells. These myeloid stromal cells are immunosuppressive, proangiogenic, and phagocytic. Elimination of these myeloid cells allowed T cells to remain active, prevented neovascularization, and prevented tumor resorption so that tumor size remained stationary. These findings show the effectiveness of adoptive CTL therapy directed against tumor stroma and open a new avenue for cancer treatments.

How a single T cell receptor recognizes both self and foreign MHC.

alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.