Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

Fluorochrome-conjugated peptide–major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin–biotin-based ‘tetramers’, can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR–pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents

Advances in T-Cell Epitope Engineering

T-cells recognize small peptide fragments (p) cradled in multiple major histocompatibility complex (MHC) molecules, termed human leukocyte antigens (HLA) in humans. These membrane-integral pMHC molecules are present on surface of all nucleated cells and allow T-cells to detect aberrant intracellular activity, be this infection with microorganisms or abnormal host biochemistry such as neoplastic division. Scanning of pMHC molecules occurs via the αβ T-cell receptor (TCR), a clonotypic, heterodimeric, and membrane-integral molecule on the T-cell surface (Miles et al., 2011a). TCRs engage pMHC molecules via six highly flexible complementarity determining region (CDR) loops and, upon productive docking with a pMHC molecule, the TCR triggers a myriad of intracellular T-cell signaling cascades (Bridgeman et al., 2012). The binding strength (or affinity) between a TCR and a cognate pMHC is relatively weak across known biological systems with monomeric “dwell times” (or half-lives) typically measured in seconds or microseconds at physiological temperatures (Miles et al., 2010; Bridgeman et al., 2012; Smith et al., 2012). This is in contrast to numerous other biological interactions such as antibodies (van der Merwe and Davis, 2003), interleukins (Morton et al., 1994), lipoproteins (Misra et al., 2001), and structural membrane proteins (Matte et al., 2012) which have half-lives measured in hours-to-days. Overall, TCR/pMHC interactions are fleeting even by the dynamic standards of cell surface interactions (van der Merwe and Davis, 2003). The evolutionary rationale for this striking functional divide can only be speculated upon but likely pertains to the primary function of T-cells. T-cells must scan large numbers of pMHC on multiple cells in series in order to identify and eliminate threats quickly. Effective immunity requires that TCR scanning time must be minimal and antigen coverage maximal. Theoretical arguments dictate that maximal immune cover of possible foreign pMHC requires each TCR to recognize huge numbers of different peptides (Mason, 1998; Sewell, 2012). This theory is now supported up by direct experimental evidence that shows a single TCR can cross-recognize millions of pMHC molecules as well or better than the native antigen (Sewell, 2012; Wooldridge et al., 2012; Ekeruche-Makinde et al., 2013). Curiously, this extensive T-cell cross-reactivity is strictly compartmentalized based on peptide length (Ekeruche-Makinde et al., 2013)

T-cell receptor-optimized peptide skewing of the T-cell repertoire can enhance antigen targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A₂₇₋₃₅ (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8+ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties

„Poesie ist das schlechte Gewissen der Literatur“. Durs Grünbeins Frankfurter Poetikvorlesung

Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this ‘ground state’ immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8+ T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment

Peptide length determines the outcome of TCR/peptide-MHCI engagement

αβ-TCRs expressed at the CD8+ T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8+ T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8+ T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8+ T-cell clonotypes

Specificity of bispecific T cell receptors and antibodies targeting peptide-HLA

Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents