Natalie A. Bowerman

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.

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.

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.

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.

Different Strategies Adopted by Kb and Ld 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 Kb and the allogeneic MHC Ld. Two distinct peptides, SIY (SIYRYYGL) and QL9 (QLSPFPFDL), act as strong and specific agonists when bound to Kb and Ld, 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 Kb revealed significant effects of three SIY residues that were clearly embedded within the Kb molecule. In contrast, QL9 binding to MHC Ld was influenced by the majority of peptide side chains, distributed across the entire length of the peptide. Binding of the SIY-Kb 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 Ld 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-Ld 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.

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-2b mice were infused with H-2k tumor cells expressing high levels of SIY peptide. Adoptively transferred 2C T cells are able to kill cross-presenting H-2b stromal cells but not H-2k 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-2k 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.

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 (KD = 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.