Faculty Opinions recommendation of Real-time analysis of T cell receptors in naive cells in vitro and in vivo reveals flexibility in synapse and signaling dynamics.

AbstractNeoantigens derived from somatic mutations have been demonstrated to correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of personalized medicine approaches although their quality and associated immune responses is not yet well understood. In a case study of metastatic malignant melanoma, we performed an in-depth characterization of neoantigens and respective T-cell responses in the context of immunotherapy with Ipilimumab. Three neoantigens identified either by immunopeptidomics or in silico prediction were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient immune repertoire recognizing these antigens. TCRs were compared in-vitro and in-vivo with multi-parametric analyses. Identified immunogenic peptides showed similar binding affinities to the human leukocyte antigen (HLA) complex and comparable differences to their wildtype counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs differed substantially in functionality and frequency. In fact, TCRs with comparably lower functional avidity and higher potential for cross-reactivity provided at least equal anti-tumor immune responses in vivo. Of note, these TCRs showed a reduced activation pattern upon primary in vitro stimulation. Exploration of the TCR-β repertoire in blood and in different tumor-related tissues over three years, offered insights on the high frequency and particular long-term persistence of low-avidity TCRs. These data indicate that qualitative differences of neoantigen-specific TCRs and their impact on function and longevity need to be considered for neoantigen targeting by adoptive T-cell therapy using TCR-transgenic T cells.Statement of translational relevanceImmunotherapy has demonstrated high efficacy in diverse malignancies. Neoantigens derived from mutations provide promising targets for safe and highly tumor-specific therapeutic approaches. Yet, single determinants of an effective and enduring T-cell mediated tumor rejection are still not well understood. We analyzed in detail seven neoantigen-specific T-cell receptors (TCRs) derived from a melanoma patient targeting three different altered peptide ligands identified by mass spectrometry and prediction analyses. Functional characterization of these TCRs revealed potent anti-tumor reactivity of all TCRs. Of special interest, TCRs with comparably lower affinity demonstrated effective in vivo activity as well as dominant spatial and temporal distribution in blood and tissue. Functional differences of TCR may require further T-cell and/or TCR engineering and should be considered for future clinical trial designs.

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H-2-restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity.

Journal of Experimental Medicine ◽

10.1084/jem.176.2.439 ◽

1992 ◽

Vol 176 (2) ◽

pp. 439-447 ◽

Cited By ~ 61

Author(s):

J L Casanova ◽

J C Cerottini ◽

M Matthes ◽

A Necker ◽

H Gournier ◽

...

Keyword(s):

T Cell ◽

T Cell Receptors ◽

Class I ◽

Antigenic Peptides ◽

Class I Mhc ◽

Cell Receptors ◽

Mhc Molecules ◽

Tcr Repertoire

We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.

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Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition

Journal of Experimental Medicine ◽

10.1084/jem.153.5.1198 ◽

1981 ◽

Vol 153 (5) ◽

pp. 1198-1214 ◽

Cited By ~ 660

Author(s):

JW Kappler ◽

B Skidmore ◽

J White ◽

P Marrack

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptors ◽

Interleukin 2 ◽

Specific Antigen ◽

Antigen Specificity ◽

Cell Receptors ◽

Cell Hybrids

We developed a method for production of antigen-specific, H-2-restricted T cell hybrids. The tumor cell partner in the fusions was itself a T cell hybrid, FS6-14.13.AG2 (or its derivatives), which could be induced to produce the growth factor, interleukin-2 (IL-2), in response to a challenge with concanavalin A, but had no known antigen specificity. The normal T cell partner in the fusions was a population of lymph node T cell blasts that had been highly enriched in antigen-specific, H-2-restricted T cells by in vivo immunization, followed by in vitro challenge with antigen and clonal expansion in IL-2-containing medium. These fusions produced hybrids that grew constitutively in culture. A sizable proportion of the hybrids demonstrated the ability to produce IL-2 in response to a challenge with specific antigen presented by irradiated spleen cells of the appropriate H-2 type. Four cloned antigen/H-2-specific hybrid lines were produced. AO-40.10 responded to chicken ovalbumin (OVA) when presented by I-A(k)-bearing cells. DC1.18.3 responded to the apo form of beef cytochrome c when presented with I-A(d). AODK-10.4 responded to keyhole limpet hemocyanin (KLH) presented with I-A (d). AODK-1.16 also responded to KLH presented by a product of the I region of H-2(d), but the data were consistent with either a product of the I-J-I-E(d) region or a combinatorial molecule with elements from both I-A(d) and I-E(d)/I-C(d). Coincidentally, AO-40.10 was shown to have an unexpected alloreactivity with a product of H-2(b) mapping to the K-I-A region. These hybrids should prove invaluable as sources of monoclonal material for the study of the receptor(s) on T cells with H-2-restricted antigen specificities. We also generated T cell hybrids with two antigen/H-2 specificities by fusing an azaguanine-resistant clone of AO-40.10 to normal T cells with a different antigen/H-2 specificity. Many of the hybrids retained reactivity to OVA plus H-2(a) and to the second antigen/H-2 combination. None reacted to either OVA plus the second H-2 type or to the second antigen plus H-2(a). One of these hybrids was successfully cloned to produce the line AOFK- 11.11.1. It retained the ability to recognize OVA plus I-A(k) inherited from one parent, and KLH plus IA(f) inherited from the other. It did not recognize OVA plus IA(f) or KLH plus I-A(k). These results have some bearing on models describing the nature of T cell receptors for antigen recognized in association with H-2 products. They do not support models in which antigen and H-2 are recognized separately by two independent T cell receptors.

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Real-Time Analysis of Apoptosis In Vivo

Methods in Cell Biology - Cytometry, 4th Edition: New Developments ◽

10.1016/s0091-679x(04)75013-x ◽

2004 ◽

pp. 343-354 ◽

Cited By ~ 3

Author(s):

Pui Lee ◽

Mark S. Segal

Keyword(s):

Real Time ◽

Time Analysis ◽

Real Time Analysis

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Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1512331113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7201-7206 ◽

Cited By ~ 41

Author(s):

Ying S. Hu ◽

Hu Cang ◽

Björn F. Lillemeier

Keyword(s):

T Cells ◽

Plasma Membrane ◽

T Cell ◽

Lymph Nodes ◽

T Cell Activation ◽

T Cell Receptors ◽

Cell Activation ◽

Cell Receptors ◽

Micrometer Scale

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

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