Hybrid structural modeling of alloantibody binding to human leukocyte antigen with rapid and reproducible cross-linking mass spectrometry.

Alloantibody recognition of donor human leukocyte antigen (HLA) is associated with poor clinical transplantation outcomes. However, the molecular and structural basis for the alloantibody-HLA interaction is not well understood. Here, we used a hybrid structural modeling approach on a previously studied alloantibody-HLA interacting pair with inputs from ab initio, in silico, and in vitro data. Highly reproducible cross-linking mass spectrometry data were obtained with both discovery- and targeted mass spectrometry-based approaches approaches. The cross-link information was then used together with predicted antibody Fv structure, predicted antibody paratope, and in silico-predicted interacting surface to model the antibody-HLA interaction. This hybrid structural modeling approach closely recapitulates the key interacting residues from a previously solved crystal structure of an alloantibody-HLA-A∗11:01 pair. These results suggest that a predictive-based hybrid structural modeling approach supplemented with cross-linking mass spectrometry data can provide functionally relevant structural models to understand the structural basis of antibody-HLA mismatch in transplantation.

Themis controls T cell activation, effector functions, and metabolism of peripheral CD8+ T cells.

Themis is important in regulating positive selection of thymocytes during T cell development, but its role in peripheral T cells is less understood. Here, we investigated T cell activation and its sequelae using a tamoxifen-mediated, acute Themis deletion mouse model. We find that proliferation, effector functions including anti-tumor killing, and up-regulation of energy metabolism are severely compromised. This study reveals the phenomenon of peripheral adaptation to loss of Themis, by demonstrating direct TCR-induced defects after acute deletion of Themis that were not evident in peripheral T cells chronically deprived of Themis in dLck-Cre deletion model. Peripheral adaptation to long-term loss was compared using chronic versus acute tamoxifen-mediated deletion and with the (chronic) dLck-Cre deletion model. We found that upon chronic tamoxifen-mediated Themis deletion, there was modulation in the gene expression profile for both TCR and cytokine signaling pathways. This profile overlapped with (chronic) dLck-Cre deletion model. Hence, we found that peripheral adaptation induced changes to both TCR and cytokine signaling modules. Our data highlight the importance of Themis in the activation of CD8+ T cells.

CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance.

Signal transduction induced by chimeric antigen receptors (CARs) is generally believed to rely on the activity of the SRC family kinase (SFK) LCK, as is the case with T cell receptor (TCR) signaling. Here, we show that CAR signaling occurs in the absence of LCK. This LCK-independent signaling requires the related SFK FYN and a CD28 intracellular domain within the CAR. LCK-deficient CAR-T cells are strongly signaled through CAR and have better in vivo efficacy with reduced exhaustion phenotype and enhanced induction of memory and proliferation. These distinctions can be attributed to the fact that FYN signaling tends to promote proliferation and survival, whereas LCK signaling promotes strong signaling that tends to lead to exhaustion. This non-canonical signaling of CAR-T cells provides insight into the initiation of both TCR and CAR signaling and has important clinical implications for improvement of CAR function.

Lymphoepithelial carcinoma of the larynx: an extremely rare tumour in a patient of Chinese descent.

Lymphoepithelial carcinoma (LEC) of the larynx is an extremely rare tumour which, unlike its nasopharyngeal counterpart, has shown a propensity to affect elderly Caucasian men and is not commonly associated with Epstein-Barr virus. We present a 70-year-old Chinese man who complained of hoarseness and dysphagia. Nasoendoscopy revealed a left supraglottic tumour. Preoperative MRI (in particular Diffusion Weighted Imaging) showed the possibility of two distinct components within a tumour. The patient underwent total pharyngolaryngectomy and bilateral selective neck dissection. The final histology report confirmed the presence of a tumour with two distinct components: predominant LEC with a smaller conventional (keratinising) squamous cell carcinoma component. The patient recovered well after surgery and subsequently underwent adjuvant radiotherapy. Final staging was pT3 N2c M0 (AJCC stage IVA). Follow-up over 2 years revealed no tumour recurrence.

Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice.

Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8+ T cell population in mice with germline Themis deficiency. Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.

Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model.

Chimeric antigen receptor T cells (CAR-T) utilize T cell receptor (TCR) signaling cascades and the recognition functions of antibodies. This allows T cells, normally restricted by the major histocompatibility complex (MHC), to be redirected to target cells by their surface antigens, such as tumor associated antigens (TAAs). CAR-T technology has achieved significant successes in treatment of certain cancers, primarily liquid cancers. Nonetheless, many challenges hinder development of this therapy, such as cytokine release syndrome (CRS) and the efficacy of CAR-T treatments for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, which has been less studied. To dissect CAR signaling, we designed a CAR that targets an epitope from latent membrane protein 2 A (LMP2 A) of the Epstein-Barr virus (EBV) presented on HLA*A02:01. Because of this, CAR and TCR signaling can be compared directly, allowing us to study the involvement of other signaling molecules, such as coreceptors. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer, but at a lower magnitude, than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. The distinct CAR signaling processes could provide explanations for clinical behavior of CAR-T therapy and suggest ways to improve the technology.

Lck bound to coreceptor is less active than free Lck.

Src family kinase Lck plays critical roles during T cell development and activation, as it phosphorylates the TCR/CD3 complex to initiate TCR signaling. Lck is present either in coreceptor-bound or coreceptor-unbound (free) forms, and we here present evidence that the two pools of Lck have different molecular properties. We discovered that the free Lck fraction exhibited higher mobility than CD8α-bound Lck in OT-I T hybridoma cells. The free Lck pool showed more activating Y394 phosphorylation than the coreceptor-bound Lck pool. Consistent with this, free Lck also had higher kinase activity, and free Lck mediated higher T cell activation as compared to coreceptor-bound Lck. Furthermore, the coreceptor-Lck coupling was independent of TCR activation. These findings give insights into the initiation of TCR signaling, suggesting that changes in coreceptor-Lck coupling constitute a mechanism for regulation of T cell sensitivity.

T cell receptor and cytokine signal integration in CD8+ T cells is mediated by the protein Themis.

T cell homeostasis and functional responsiveness require signals from self-peptide-major histocompatibility complex (self-pMHC) and cytokines, but the mechanisms controlling this signal integration are unknown. Using a conditional deletion of the T cell lineage-specific protein Themis, we show that Themis is required for the maintenance of peripheral CD8+ T cells and for proliferative CD8+ T cell responses to low-affinity pMHC aided by cytokines. Themis-deficient peripheral T cells show a phenotype indicative of reduced tonic signaling from self-pMHC, strongly suggesting that Themis is a positive regulator of T cell receptor signal strength in response to low-affinity self-pMHC in peripheral T cells. Signals from low-affinity pMHC and cytokines synergistically induce phosphorylation of the kinase Akt, metabolic changes and c-Myc transcription factor induction in CD8+ T cells only in the presence of Themis. This function of Themis is mediated through Shp1 phosphatase, as peripheral Themis and Shp1 double deletion rescues the peripheral CD8+ T cell maintenance.

Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01.

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.

Themis-associated phosphatase activity controls signaling in T cell development.

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.

Nonstimulatory peptide-MHC enhances human T-cell antigen-specific responses by amplifying proximal TCR signaling.

Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide-MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.

Design and validation of conditional ligands for HLA-B*08:01, HLA-B*15:01, HLA-B*35:01, and HLA-B*44:05.

We designed conditional ligands restricted to HLA-B*08:01, -B*35:01, and -B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context.

Bioorthogonal cleavage and exchange of major histocompatibility complex ligands by employing azobenzene-containing peptides.

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.