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TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

ABSTRACT: The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide-major histocompatibility complex (pMHC) molecules on other cells. If productive, these interactions promote the formation of an immunological synapse. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence resonance energy transfer (FRET) between fluorescently tagged TCRs and their cognate pMHC ligands to measure the kinetics of TCR-pMHC binding in situ. When compared with solution measurements, the dissociation of this complex was increased significantly (4-12-fold). Disruption of actin polymers reversed this effect, indicating that cytoskeletal dynamics destabilize this interaction directly or indirectly. Nevertheless, TCR affinity for pMHC was significantly elevated as the result of a large (about 100-fold) increase in the association rate, a likely consequence of complementary molecular orientation and clustering. In helper T cells, the CD4 molecule has been proposed to bind cooperatively with the TCR to the same pMHC complex. However, CD4 blockade had no effect on the synaptic TCR affinity, nor did it destabilize TCR-pMHC complexes, indicating that the TCR binds pMHC independently of CD4.

SUBMITTER: Huppa JB

PROVIDER: S-EPMC3273423 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

Huppa Johannes B JB Axmann Markus M Mörtelmaier Manuel A MA Lillemeier Björn F BF Newell Evan W EW Brameshuber Mario M Klein Lawrence O LO Schütz Gerhard J GJ Davis Mark M MM

Nature 20100201 7283

The recognition of foreign antigens by T lymphocytes is essential to most adaptive immune responses. It is driven by specific T-cell antigen receptors (TCRs) binding to antigenic peptide-major histocompatibility complex (pMHC) molecules on other cells. If productive, these interactions promote the formation of an immunological synapse. Here we show that synaptic TCR-pMHC binding dynamics differ significantly from TCR-pMHC binding in solution. We used single-molecule microscopy and fluorescence r ...[more]

PMID: 20164930

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Project description:BackgroundAffinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, but also bear substantial risks for off-target toxicities. Most preclinical studies have focused on T cell responses to antigen-specific stimulation. In contrast, little is known on the regulation of T cell responsiveness through continuous TCR triggering and consequent tonic signaling. Here, we addressed the question whether increasing the TCR affinity can lead to chronic interactions occurring directly between TCRs and MHC-(self) molecules, which may modulate the overall functional potency of tumor-redirected CD8 T cells. For this purpose, we developed two complementary human CD8 T cell models (i.e. HLA-A2 knock-in and knock-out) engineered with incremental-affinity TCRs to the HLA-A2/NY-ESO-1 tumor antigen.MethodsThe impact of HLA-A2 recognition, depending on TCR affinity, was assessed at the levels of the TCR/CD3 complex, regulatory receptors, and signaling, under steady-state conditions and in kinetic studies. The quality of CD8 T cell responses was further evaluated by gene expression and multiplex cytokine profiling, as well as real-time quantitative cell killing, combined with co-culture assays.ResultsWe found that HLA-A2 per se (in absence of cognate peptide) can trigger chronic activation followed by a tolerance-like state of tumor-redirected CD8 T cells with increased-affinity TCRs. HLA-A2pos but not HLA-A2neg T cells displayed an activation phenotype, associated with enhanced upregulation of c-CBL and multiple inhibitory receptors. T cell activation preceded TCR/CD3 downmodulation, impaired TCR signaling and functional hyporesponsiveness. This stepwise activation-to-hyporesponsive state was dependent on TCR affinity and already detectable at the upper end of the physiological affinity range (KD ≤ 1 μM). Similar findings were made when affinity-increased HLA-A2neg CD8 T cells were chronically exposed to HLA-A2pos-expressing target cells.ConclusionsOur observations indicate that sustained interactions between affinity-increased TCR and self-MHC can directly adjust the functional potential of T cells, even in the absence of antigen-specific stimulation. The observed tolerance-like state depends on TCR affinity and has therefore potential implications for the design of affinity-improved TCRs for adoptive T cell therapy, as several engineered TCRs currently used in clinical trials share similar affinity properties.

Project description:Recruitment and activation of CD8 T cells occur through specific triggering of T cell receptor (TCR) by peptide-bound human leucocyte antigen (HLA) ligands. Within the generated trimeric TCR-peptide:HLA complex, the molecular binding affinities between peptide and HLA, and between TCR and peptide:HLA both impact T cell functional outcomes. However, how their individual and combined effects modulate immunogenicity and overall T cell responsiveness has not been investigated systematically. Here, we established two panels of human tumor peptide variants differing in their affinity to HLA. For precise characterization, we developed the "blue peptide assay", an upgraded cell-based approach to measure the peptide:HLA affinity. These peptide variants were then used to investigate the cross-reactivity of tumor antigen-specific CD8 T cell clonotypes derived from blood of cancer patients after vaccination with either the native or an affinity-optimized Melan-A/MART-1 epitope, or isolated from tumor infiltrated lymph nodes (TILNs). Vaccines containing the native tumor epitope generated T cells with better functionality, and superior cross-reactivity against potential low affinity escape epitopes, as compared to T cells induced by vaccines containing an HLA affinity-optimized epitope. Comparatively, Melan-A/MART-1-specific TILN cells displayed functional and cross-reactive profiles that were heterogeneous and clonotype-dependent. Finally, we took advantage of a collection of T cells expressing affinity-optimized NY-ESO-1-specific TCRs to interrogate the individual and combined impact of peptide:HLA and TCR-pHLA affinities on overall CD8 T cell responses. We found profound and distinct effects of both biophysical parameters, with additive contributions and absence of hierarchical dominance. Altogether, the biological impact of peptide:HLA and TCR-pHLA affinities on T cell responses was carefully dissected in two antigenic systems, frequently targeted in human cancer immunotherapy. Our technology and stepwise comparison open new insights into the rational design and selection of vaccine-associated tumor-specific epitopes and highlight the functional and cross-reactivity profiles that endow T cells with best tumor control capacity.

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TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

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TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.

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