Identifying a minor histocompatibility antigen in mauritian cynomolgus macaques encoded by APOBEC3C

Allogeneic hematopoietic stem cell transplants can lead to dramatic reductions in human immunodeficiency virus (HIV) reservoirs. This effect is partially mediated by donor T cells recognizing lymphocyte-expressed minor histocompatibility antigens (mHAgs). The potential to mark malignant and latently infected cells for destruction makes mHAgs attractive targets for cellular immunotherapies. However, testing such HIV reservoir reduction strategies will likely require preclinical studies in non-human primates (NHPs). In this study, we used a combination of alloimmunization, whole exome sequencing, and bioinformatics to identify an mHAg in Mauritian cynomolgus macaques (MCMs). We mapped the minimal optimal epitope to a 10-mer peptide (SW10) in apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3C (APOBEC3C) and determined the major histocompatibility complex class I restriction element as Mafa-A1∗063, which is expressed in almost 90% of MCMs. APOBEC3C SW10-specific CD8+ T cells recognized immortalized B cells but not fibroblasts from an mHAg-positive MCM. These results provide a framework for identifying mHAgs in a non-transplant setting and suggest that APOBEC3C SW10 could be used as a model antigen to test mHAg-targeted therapies in NHPs

CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity

CD8+ T-­cell specificity is compromised at a defined major histocompatibility complex class I/CD8 affinity threshold

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity

Divergent roles for antigenic drive in the aetiology of primary versus dasatinib-associated CD8(+) TCR-Vβ(+) expansions

CD8(+) T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8(+) T-cell receptor (TCR)-V beta(+) populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-V beta(+)) and residual (TCR-V beta(-)) CD8(+) T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8(+) TCR-V beta(+) expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8(+) TCR-V beta(+) expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8(+) TCR-V beta(+) expansions.Peer reviewe

CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold

The CD8 coreceptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR) binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~ 1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~ 10-fold). In this study, we used a panel of MHCI mutants with altered CD8 binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity. The pMHCI/CD8 interaction controls specificit

TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection

Objective Chronic hepatitis B virus (HBV) infection is characterised by HBV-specific CD8+ T cell dysfunction that has been linked to Tcell exhaustion, a distinct differentiation programme associated with persisting antigen recognition. Recently, Thymocyte Selection-Associated High Mobility Group Box (TOX) was identified as master regulator of CD8+ T cell exhaustion. Here, we addressed the role of TOX in HBV-specific CD8+ T cell dysfunction associated with different clinical phases of infection. Design We investigated TOX expression in HBV-specific CD8+ T cells from 53 HLA-A*01:01, HLA-A*11:01 and HLA-A*02:01 positive patients from different HBV infection phases and compared it to hepatitis C virus (HCV)-specific, cytomegalovirus (CMV)-specific, Epstein-Barr virus (EBV)-specific and influenza virus (FLU)-specific CD8+ T cells. Phenotypic and functional analyses of virus-specific CD8+ T cells were performed after peptide-loaded tetramer-enrichment and peptide-specific expansion. Results Our results show that TOX expression in HBV-specific CD8+ T cells is linked to chronic antigen stimulation, correlates with viral load and is associated with phenotypic and functional characteristics of T-cell exhaustion. In contrast, similar TOX expression in EBV-specific and CMV-specific CD8+ T cells is not linked to T-cell dysfunction suggesting different underlying programmes. TOX expression in HBV-specific CD8+ T cells is also affected by targeted antigens, for example, core versus polymerase. In HBV-specific CD8+ T cells, TOX expression is maintained after spontaneous or therapy-mediated viral control in chronic but not self-limiting acute HBV infection indicating a permanent molecular imprint after chronic but not temporary stimulation. Conclusion Our data highlight TOX as biomarker specific for dysfunctional virus-specific CD8+ T cells in the context of an actively persisting infection

Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching’ can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases

Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load

Objective A hallmark of chronic HBV (cHBV) infection is the presence of impaired HBV-specific CD8+ T cell responses. Functional T cell exhaustion induced by persistent antigen stimulation is considered a major mechanism underlying this impairment. However, due to their low frequencies in chronic infection, it is currently unknown whether HBV-specific CD8+ T cells targeting different epitopes are similarly impaired and share molecular profiles indicative of T cell exhaustion. Design By applying peptide-loaded MHC I tetramer-based enrichment, we could detect HBV-specific CD8+ T cells targeting epitopes in the HBV core and the polymerase proteins in the majority of 85 tested cHBV patients with low viral loads. Lower detection rates were obtained for envelope-specific CD8+ T cells. Subsequently, we performed phenotypic and functional in-depth analyses. Results HBV-specific CD8+ T cells are not terminally exhausted but rather exhibit a memory-like phenotype in patients with low viral load possibly reflecting weak ongoing cognate antigen recognition. Moreover, HBV-specific CD8+ T cells targeting core versus polymerase epitopes significantly differed in frequency, phenotype and function. In particular, in comparison with core-specific CD8+ T cells, a higher frequency of polymerase-specific CD8+ T cells expressed CD38, KLRG1 and Eomes accompanied by low T-bet expression and downregulated CD127 indicative of a more severe T cell exhaustion. In addition, polymerase-specific CD8+ T cells exhibited a reduced expansion capacity that was linked to a dysbalanced TCF1/BCL2 expression. Conclusions Overall, the molecular mechanisms underlying impaired T cell responses differ with respect to the targeted HBV antigens. These results have potential implications for immunotherapeutic approaches in HBV cure

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1‐specific CD8+ T cells

Objectives Vaccines that prime Wilms' tumor 1 (WT1)‐specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C‐type lectin‐like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T‐cell responses. We developed a new vaccine comprising a human anti‐CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1‐specific CD8+ T cells. Methods WT1 was genetically fused to antibodies specific for human CLEC9A, DEC‐205 or β‐galactosidase (untargeted control). Activation of WT1‐specific CD8+ T‐cell lines following cross‐presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1‐specific CD8+ T cells, were used to investigate naïve WT1‐specific CD8+ T‐cell priming. Results The CLEC9A‐WT1 vaccine promoted cross‐presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC‐205‐WT1 and untargeted control‐WT1 vaccines. Conclusions Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag‐presenting cells via DEC‐205, suggesting that cross‐presentation by CD141+ DCs is sufficient for effective CD8+ T‐cell priming in humans. The CLEC9A‐WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1

Targeted suppression of autoreactive CD8+ T-cell activation using blocking anti-CD8 antibodies

CD8+ T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8+ T-cell population are not desirable because the associated lack of speci city can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8+ T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-speci c CD8+ T-cells are relatively CD8-independent. These generic di erences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-speci c TCRs according to the monomeric interaction a nity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, “blocking” anti-CD8 antibodies can suppress autoreactive CD8+ T-cell activation in a relatively selective manner. These ndings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8+ T-cell compartment