All Publications

  • Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides. Nature Yang, X., Garner, L. I., Zvyagin, I. V., Paley, M. A., Komech, E. A., Jude, K. M., Zhao, X., Fernandes, R. A., Hassman, L. M., Paley, G. L., Savvides, C. S., Brackenridge, S., Quastel, M. N., Chudakov, D. M., Bowness, P., Yokoyama, W. M., McMichael, A. J., Gillespie, G. M., Garcia, K. C. 2022


    Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region-complementary-determining region 3β (BV9-CDR3β) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

    View details for DOI 10.1038/s41586-022-05501-7

    View details for PubMedID 36477533

  • Localized ablative immunotherapy drives de novo CD8+ T-cell responses to poorly immunogenic tumors. Journal for immunotherapy of cancer Hoover, A. R., Kaabinejadian, S., Krawic, J. R., Sun, X., Naqash, A. R., Yin, Q., Yang, X., Christopher Garcia, K., Davis, M. M., Hildebrand, W. H., Chen, W. R. 2022; 10 (10)


    BACKGROUND: Localized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns to stimulate T-cell immunity, but its immune stimulating effect is limited, particularly against metastatic tumors.METHODS: In this study, we combined photothermal therapy with a potent immune stimulant, N-dihydrogalactochitosan, to create a local ablative immunotherapy which we refer to as laser immunotherapy (LIT). Mice bearing B16-F10 tumors were treated with LIT when the tumors reached 0.5 cm3 and were monitored for survival, T-cell activation, and the ability to resist tumor rechallenge.RESULTS: We found that LIT stimulated a stronger and more consistent antitumor T-cell response to the immunologically 'cold' B16-F10 melanoma tumors and conferred a long-term antitumor memory on tumor rechallenge. Furthermore, we discovered that LIT generated de novo CD8+ T-cell responses that strongly correlated with animal survival and tumor rejection.CONCLUSION: In summary, our findings demonstrate that LIT enhances the activation of T cells and drives de novo antitumor T-cell responses. The data presented herein suggests that localized ablative immunotherapies have great potential to synergize with immune checkpoint therapies to enhance its efficacy, resulting in improved antitumor immunity.

    View details for DOI 10.1136/jitc-2022-004973

    View details for PubMedID 36253002

  • Tuning T cell receptor sensitivity through catch bond engineering. Science (New York, N.Y.) Zhao, X., Kolawole, E. M., Chan, W., Feng, Y., Yang, X., Gee, M. H., Jude, K. M., Sibener, L. V., Fordyce, P. M., Germain, R. N., Evavold, B. D., Garcia, K. C. 2022; 376 (6589): eabl5282


    Adoptive cell therapy using engineered T cell receptors (TCRs) is a promising approach for targeting cancer antigens, but tumor-reactive TCRs are often weakly responsive to their target ligands, peptide-major histocompatibility complexes (pMHCs). Affinity-matured TCRs can enhance the efficacy of TCR-T cell therapy but can also cross-react with off-target antigens, resulting in organ immunopathology. We developed an alternative strategy to isolate TCR mutants that exhibited high activation signals coupled with low-affinity pMHC binding through the acquisition of catch bonds. Engineered analogs of a tumor antigen MAGE-A3-specific TCR maintained physiological affinities while exhibiting enhanced target killing potency and undetectable cross-reactivity, compared with a high-affinity clinically tested TCR that exhibited lethal cross-reactivity with a cardiac antigen. Catch bond engineering is a biophysically based strategy to tune high-sensitivity TCRs for T cell therapy with reduced potential for adverse cross-reactivity.

    View details for DOI 10.1126/science.abl5282

    View details for PubMedID 35389803

  • T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes. Nature biotechnology Ali, M., Giannakopoulou, E., Li, Y., Lehander, M., Virding Culleton, S., Yang, W., Knetter, C., Odabasi, M. C., Bollineni, R. C., Yang, X., Foldvari, Z., Boschen, M., Taraldsrud, E., Stronen, E., Toebes, M., Hillen, A., Mazzi, S., de Ru, A. H., Janssen, G. M., Kolstad, A., Tjonnfjord, G. E., Lie, B. A., Griffioen, M., Lehmann, S., Osnes, L. T., Buechner, J., Garcia, K. C., Schumacher, T. N., van Veelen, P. A., Leisegang, M., Jacobsen, S. E., Woll, P., Olweus, J. 2021


    Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that Tcells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific Tcells. TCR-modified Tcells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.

    View details for DOI 10.1038/s41587-021-01089-x

    View details for PubMedID 34873326

  • Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity Chiou, S. H., Tseng, D. n., Reuben, A. n., Mallajosyula, V. n., Molina, I. S., Conley, S. n., Wilhelmy, J. n., McSween, A. M., Yang, X. n., Nishimiya, D. n., Sinha, R. n., Nabet, B. Y., Wang, C. n., Shrager, J. B., Berry, M. F., Backhus, L. n., Lui, N. S., Wakelee, H. A., Neal, J. W., Padda, S. K., Berry, G. J., Delaidelli, A. n., Sorensen, P. H., Sotillo, E. n., Tran, P. n., Benson, J. A., Richards, R. n., Labanieh, L. n., Klysz, D. D., Louis, D. M., Feldman, S. A., Diehn, M. n., Weissman, I. L., Zhang, J. n., Wistuba, I. I., Futreal, P. A., Heymach, J. V., Garcia, K. C., Mackall, C. L., Davis, M. M. 2021; 54 (3): 586–602.e8


    To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRβ chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

    View details for DOI 10.1016/j.immuni.2021.02.014

    View details for PubMedID 33691136

  • Discovery of surrogate agonists for visceral fat Treg cells that modulate metabolic indices in vivo. eLife Fernandes, R. A., Li, C., Wang, G., Yang, X., Savvides, C. S., Glassman, C. R., Dong, S., Luxenberg, E., Sibener, L. V., Birnbaum, M. E., Benoist, C., Mathis, D., Garcia, K. C. 2020; 9


    T regulatory (Treg) cells play vital roles in modulating immunity and tissue homeostasis. Their actions depend on TCR recognition of peptide-MHC molecules; yet the degree of peptide specificity of Treg-cell function, and whether Treg ligands can be used to manipulate Treg cell biology are unknown. Here, we developed an Ab-peptide library that enabled unbiased screening of peptides recognized by a bona fide murine Treg cell clone isolated from the visceral adipose tissue (VAT), and identified surrogate agonist peptides, with differing affinities and signaling potencies. The VAT-Treg cells expanded in vivo by one of the surrogate agonists preserved the typical VAT-Treg transcriptional programs. Immunization with this surrogate, especially when coupled with blockade of TNFa signaling, expanded VAT-Treg cells, resulting in protection from inflammation and improved metabolic indices, including promotion of insulin sensitivity. These studies suggest that antigen-specific targeting of VAT-localized Treg cells could eventually be a strategy for improving metabolic disease.

    View details for DOI 10.7554/eLife.58463

    View details for PubMedID 32773038

  • Discovery of a novel shared tumor antigen in human lung cancer. Tseng, D., Chiou, S., Yang, X., Reuben, A., Wilhelmy, J., McSween, A., Conley, S., Sinha, R., Nabet, B., Wang, C., Shrager, J. B., Berry, M. F., Backhus, L., Lui, n., Wakelee, H. A., Neal, J. W., Zhang, J., Garcia, K., Mackall, C., Davis, M. AMER SOC CLINICAL ONCOLOGY. 2020
  • Towards the identification of novel tumor antigens in human lung cancer. Chiou, S., Tseng, D., Wang, C., Reuben, A., Yang, X., Wilhelmy, J., McSween, A., Zhang, J., Shrager, J., Garcia, K., Davis, M. AMER ASSOC CANCER RESEARCH. 2020: 44–45
  • Immune receptor inhibition through enforced phosphatase recruitment. Nature Fernandes, R. A., Su, L. n., Nishiga, Y. n., Ren, J. n., Bhuiyan, A. M., Cheng, N. n., Kuo, C. J., Picton, L. K., Ohtsuki, S. n., Majzner, R. G., Rietberg, S. P., Mackall, C. L., Yin, Q. n., Ali, L. R., Yang, X. n., Savvides, C. S., Sage, J. n., Dougan, M. n., Garcia, K. C. 2020


    Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

    View details for DOI 10.1038/s41586-020-2851-2

    View details for PubMedID 33087934

  • Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding. Cell Sibener, L. V., Fernandes, R. A., Kolawole, E. M., Carbone, C. B., Liu, F., McAffee, D., Birnbaum, M. E., Yang, X., Su, L. F., Yu, W., Dong, S., Gee, M. H., Jude, K. M., Davis, M. M., Groves, J. T., Goddard, W. A., Heath, J. R., Evavold, B. D., Vale, R. D., Garcia, K. C. 2018; 174 (3): 672


    TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human Tcell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds withinthe TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

    View details for PubMedID 30053426

  • Stress-testing the relationship between T cell receptor/peptide-MHC affinity and cross-reactivity using peptide velcro. Proceedings of the National Academy of Sciences of the United States of America Gee, M. H., Sibener, L. V., Birnbaum, M. E., Jude, K. M., Yang, X., Fernandes, R. A., Mendoza, J. L., Glassman, C. R., Garcia, K. C. 2018


    T cell receptors (TCRs) bind to peptide-major histocompatibility complex (pMHC) with low affinity (Kd muM), which is generally assumed to facilitate cross-reactive TCR "scanning" of ligands. To understand the relationship between TCR/pMHC affinity and cross-reactivity, we sought to engineer an additional weak interaction, termed "velcro," between the TCR and pMHC to probe the specificities of TCRs at relatively low and high affinities. This additional interaction was generated through an eight-amino acid peptide library covalently linked to the N terminus of the MHC-bound peptide. Velcro was selected through an affinity-based isolation and was subsequently shown to enhance the cognate TCR/pMHC affinity in a peptide-dependent manner by 10-fold. This was sufficient to convert a nonstimulatory ultra-low-affinity ligand into a stimulatory ligand. An X-ray crystallographic structure revealed how velcro interacts with the TCR. To probe TCR cross-reactivity, we screened TCRs against yeast-displayed pMHC libraries with and without velcro, and found that the peptide cross-reactivity profiles of low-affinity (Kd > 100 muM) and high-affinity (Kd muM) TCR/pMHC interactions are remarkably similar. The conservation of recognition of the TCR for pMHC across affinities reveals the nature of low-affinity ligands for which there are important biological functions and has implications for understanding the specificities of affinity-matured TCRs.

    View details for PubMedID 30021852

  • Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes CELL Gee, M. H., Han, A., Lofgren, S. M., Beausang, J. F., Mendoza, J. L., Birnbaum, M. E., Bethune, M. T., Fischer, S., Yang, X., Gomez-Eerland, R., Bingham, D. B., Sibener, L. V., Fernandes, R. A., Velasco, A., Baltimore, D., Schumacher, T. N., Khatri, P., Quake, S. R., Davis, M. M., Garcia, K. 2018; 172 (3): 549-+


    The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A∗02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

    View details for PubMedID 29275860

    View details for PubMedCentralID PMC5786495

  • Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy JOURNAL OF EXPERIMENTAL MEDICINE Chheda, Z. S., Kohanbash, G., Okada, K., Jahan, N., Sidney, J., Pecoraro, M., Yang, X., Carrera, D. A., Downey, K. M., Shrivastav, S., Liu, S., Lin, Y., Lagisetti, C., Chuntova, P., Watchmaker, P. B., Mueller, S., Pollack, I. F., Rajalingam, R., Carcaboso, A. M., Mann, M., Sette, A., Garcia, K., Hou, Y., Okada, H. 2018; 215 (1): 141–57


    The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

    View details for PubMedID 29203539

    View details for PubMedCentralID PMC5748856