Background Epstein Barr virus (EBV) is a highly prevalent herpesvirus worldwide and its infection is associated with malignancies such as nasopharyngeal cancer (NPC), gastric cancer, lymphoepithelioma cancer and lymphoproliferative disorders. Virus-specific T (VST) cell therapies using T cells reactive against EBV have shown limited efficacy in clinical trials with several practical limitations such as the long preparation period and the variations of pre-existing EBV-reactive T cells in different donors. To overcome these technical constraints of the VST approach, we have established the genetically engineered T cells targeting EBV antigens with lentiviral transduction. A proof-of-concept clinical validation was further conducted against EBV+ malignancies (trial No. ChiCTR2100044497). Methods T cells were activated by CD3/CD28 stimulation, lentivirally transduced with codon-optimized CAR (targeting EBV envelope protein) or TCR (targeting EBV latent membrane protein) and further expanded ex vivo. Transgene expression and the T cell phenotype were assessed by flow cytometry. In vitro anti-tumor efficacy was analyzed by IFN-γ ELISA and killing assays such as RTCA Xcelligence. In vivo, the antitumor efficacy of EBV-specific T cells was evaluated in immunocompromised mice bearing human tumor xenografts. Anti-tumor responses in lymphoma and NPC were evaluated by Lugano 2014 and RECIST 1.1 respectively. Results T cells lentivirally transduced with EBV antigen-specific CAR or TCR demonstrated satisfactory surface expression of the introduced receptor and showed increased cytokine production when stimulated to target expressing tumor cells in vitro (figure 1). We have identified the TCRs against EBV latent membrane protein with high functional avidity as determined by peptide dose-response functional assays. In vitro cytotoxicity assays demonstrated strong killing of antigen positive tumor cell lines of the engineered EBV-specific CAR-T or TCR-T cells, while sparing the antigen-negative cells, indicating the specific T cell response. The single intravenous infusion of engineered EBV-specific T cells also led to dose-dependent anti-tumor responses in immunocompromised mouse xenograft models (figure 1). In the clinical trial, two patients with lymphoproliferative disease and one patient with advanced NPC treated with engineered EBV-targeting T cells experienced sustained objective responses. Moreover, in the NPC patient, the EBV DNA copy number decreased from 3.32*103 copies/ml at baseline to normal (<5.00*102 copies/ml) 2 months post infusion. The infused cells also demonstrated satisfactory safety profile with no neurotoxicity and ≥ Grade 2 cytokine release syndrome observed. Conclusions Our data demonstrate the feasibility of using genetically engineered T cells targeting EBV antigens such as envelope protein or latent membrane protein to treat EBV-associated malignancies. Trial Registration Chinese Clinical Trial Registry: ChiCTR2100044497 Ethics Approval All animal studies were conducted at Guangzhou Regenerative Medicine and Health, Guangdong Laboratory as approved by IACUC (2020125). The clinical study was approved by the first affiliated hospital of Zhengzhou University's Ethics Board (L2021-Y027–005).

Next‐generation sequencing technologies have revealed that adaptive immunity is underpinned by a vast array of T cell receptors (TCRs). However, it has proven difficult to interpret these extensive datasets, in part because the field previously lacked a comprehensive and internally controlled reference atlas encompassing the full spectrum of phenotypically defined subsets in each lineage. To address this knowledge gap, we sequenced 74 million TCRs expressed by discrete CD4+ and CD8+ memory T cell populations across genetically unrelated individuals, providing a resource to inform basic and applied studies of repertoire compartmentalisation within the adaptive immune system. Using this resource, we found that T cell differentiation could not be explained solely by the self‐renewing effector model and, unexpectedly, that T cell fate could be predicted by specific genetic and physicochemical features of the TCR.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promising potential for opening new avenues in regenerative medicine. However, since the tumorigenic potential of undifferentiated pluripotent stem cells (PSCs) is a major safety concern for clinical transplantation, inducible Caspase-9 (iC9) is under consideration for use as a fail-safe system. Here, we used targeted gene editing to introduce the iC9 system into human iPSCs, and then interrogated the efficiency of inducible apoptosis with normal iPSCs as well as diseased iPSCs derived from patients with acute myeloid leukemia (AML-iPSCs). The iC9 system induced quick and efficient apoptosis to iPSCs in vitro. More importantly, complete eradication of malignant cells without AML recurrence was shown in disease mouse models by using AML-iPSCs. In parallel, it shed light on several limitations of the iC9 system usage. Our results suggest that careful use of the iC9 system will serve as an important countermeasure against posttransplantation adverse events in stem cell transplantation therapies.

During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ0 mice. We found that CD3δ0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome.

Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic "mimics" using subunits that do not exist in the natural world. We developed a platform based on D-amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus-specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.

BACKGROUND Increasing evidence indicates a role for EBV in the pathogenesis of multiple sclerosis (MS). EBV-infected autoreactive B cells might accumulate in the CNS because of defective cytotoxic CD8+ T cell immunity. We sought to determine the feasibility and safety of treating progressive MS patients with autologous EBV-specific T cell therapy. METHODS An open-label phase I trial was designed to treat 5 patients with secondary progressive MS and 5 patients with primary progressive MS with 4 escalating doses of in vitro-expanded autologous EBV-specific T cells targeting EBV nuclear antigen 1, latent membrane protein 1 (LMP1), and LMP2A. Following adoptive immunotherapy, we monitored the patients for safety and clinical responses. RESULTS Of the 13 recruited participants, 10 received the full course of T cell therapy. There were no serious adverse events. Seven patients showed improvement, with 6 experiencing both symptomatic and objective neurological improvement, together with a reduction in fatigue, improved quality of life, and, in 3 patients, reduced intrathecal IgG production. All 6 patients receiving T cells with strong EBV reactivity showed clinical improvement, whereas only 1 of the 4 patients receiving T cells with weak EBV reactivity showed improvement (P = 0.033, Fisher's exact test). CONCLUSION EBV-specific adoptive T cell therapy was well tolerated. Clinical improvement following treatment was associated with the potency of EBV-specific reactivity of the administered T cells. Further clinical trials are warranted to determine the efficacy of EBV-specific T cell therapy in MS. TRIAL REGISTRATION Australian New Zealand Clinical Trials Registry, ACTRN12615000422527. FUNDING MS Queensland, MS Research Australia, Perpetual Trustee Company Ltd., and donations from private individuals who wish to remain anonymous.

Mounting evidence indicates that infection with Epstein–Barr virus (EBV) has a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8 + T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T-cell response to EBV in MS, using flow cytometry and intracellular IFN-γ staining to measure T-cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of human leukocyte antigen (HLA)-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus (CMV), in 95 patients and 56 EBV-seropositive healthy subjects. In 20 HLA-A2 + healthy subjects and 20 HLA-A2 + patients we also analysed CD8 + T cells specific for individual peptides, measured by binding to HLA-peptide complexes and production of IFN-γ, TNF-α and IL-2. We found a decreased CD8 + T-cell response to EBV lytic, but not CMV lytic, antigens at the onset of MS and at all subsequent disease stages. CD8 + T cells directed against EBV latent antigens were increased but had reduced cytokine polyfunctionality indicating T-cell exhaustion. During attacks the EBV-specific CD4 + and CD8 + T-cell populations expanded, with increased functionality of latent-specific CD8 + T cells. With increasing disease duration, EBV-specific CD4 + and CD8 + T cells progressively declined, consistent with T-cell exhaustion. The anti-EBNA1 IgG titre correlated inversely with the EBV-specific CD8 + T-cell frequency. We postulate that defective CD8 + T-cell control of EBV reactivation leads to an expanded population of latently infected cells, including autoreactive B cells.

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.

Autoimmune diseases such as type I diabetes (TID) and multiple sclerosis (MS) are chronic conditions that have a significant impact on quality of life. CD8+ T-cells play an important role in the pathogenesis of these diseases. However, drugs that target the entire CD8+ T-cell population are not desirable because this population provides protection against infection. Accordingly, there is an urgent requirement to develop novel treatment strategies that exclusively target the autoreactive CD8+ T-cell population. CD8+ T-cells express a molecule called CD8 at their cell surface which assists with activation. We have demonstrated that autoreactive CD8+ T-cells are entirely dependent on CD8 for activation. In stark contrast, pathogen specific CD8+ T-cells are characterized by higher functional sensitivity and are relatively CD8 independent. This represents an intrinsic difference that can be exploited for therapeutic benefit. Our data suggests that “blocking anti-CD8 antibodies” can be used to block autoreactive CD8+ T-cell attack without affecting pathogen specific immunity, a key goal in the treatment of autoimmunity. The generation of “blocking anti-CD8 antibodies” offers an opportunity to develop a novel therapeutic approach that can be used to treat CD8+ T-cell mediated autoimmunity. In order to conduct a further assessment, we are currently developing a strategy for in vivo validation of the approach.

Genetic variability greatly impacts on the human immune system and is the basis for inter-patient variability in alloimmune responses and allograft rejection. While human leukocyte antigen (HLA) polymorphism clearly has a dominant impact on the cellular immune response, we investigated if allelic sequence variation within T-cell receptor (TCR) loci represents another genetic variable influencing the T-cell alloresponse. Based on previous reports that some variable (V) gene polymorphisms encode for TCR regions that make contact with the peptide-HLA complex, a pyrosequencing technique was used to determine whether T-cells expressing certain TCR V gene alleles preferentially responded to HLA-mismatched stimulator cells. Frequently occurring polymorphisms within both TCR-α and -ß gene segments were shown to significantly influence T cell responsiveness to cells expressing a single mismatched HLA allele. The most striking bias was observed with TRAV8-7 and TRBV9, whereby individuals who were heterozygous for the two alleles responded to particular alloantigens with over 95% of T cells expressing only one of the alleles. Thus, polymorphism in the TCR loci is another genetic variable that may contribute toward inter-individual variability in T cell alloresponses and allograft rejection.

[This corrects the article DOI: 10.1038/cti.2016.87.].

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.

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 pMHCl/CD8 interaction (similar to 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 pMHCl/CD8 affinity (similar to 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 pMHCl/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 nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B 2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1 2/2 clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1 2/2 clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells. M ucosal-associated invariant T (MAIT) cells are the most abundant nonconventional T cell subset, accounting for up to 5% of all T cells in humans, and are thought to be important for the control of a number of bacterial, fungal, and yeast infections (1–5). These so-called innate-like T cells, which are mostly found in the blood, the liver, and at mucosal surfaces, express a semi-invariant TCR consisting of an a-chain using the canonical TRAV1-2–TRAJ33/12/20 (Va7.2-Ja33/12/20) rearrangements (6). MAIT cells acquire effector functions during thymic selection and readily respond to Ags derived from many (but not all) bacteria such as Escherichia coli, Klebsiella pneu-moniae, Mycobacterium tuberculosis, or Staphylococcus epider-mis as well as several yeast species in the periphery without prior priming (3, 7). MAIT cell activation is mediated by the interaction between the TCR and microbe-derived Ags presented by the nonclassical MHC-related protein 1 (MR1) and results in the secretion of cytokines as well as in granzyme-and …

ABSTRACT Reconstitution of T cell immunity is absolutely critical for the effective control of virus-associated infectious complications in hematopoietic stem cell transplant (HSCT) recipients. Coinfection with genetic variants of human cytomegalovirus (CMV) in transplant recipients has been linked to clinical disease manifestation; however, how these genetic variants impact T cell immune reconstitution remains poorly understood. In this study, we have evaluated dynamic changes in the emergence of genetic variants of CMV in HSCT recipients and correlated these changes with reconstitution of antiviral T cell responses. In an analysis of single nucleotide polymorphisms within sequences encoding HLA class I-restricted CMV epitopes from the immediate early 1 gene of CMV, coinfection with genetically distinct variants of CMV was detected in 52% of patients. However, in spite of exposure to multiple viral variants, the T cell responses in these patients were preferentially directed to a limited repertoire of HLA class I-restricted CMV epitopes, either conserved, variant, or cross-reactive. More importantly, we also demonstrate that long-term control of CMV infection after HSCT is primarily mediated through the efficient induction of stable antiviral T cell immunity irrespective of the nature of the antigenic target. These observations provide important insights for the future design of antiviral T cell-based immunotherapeutic strategies for transplant recipients, emphasizing the critical impact of robust immune reconstitution on efficient control of viral infection. IMPORTANCE Infection and disease caused by human cytomegalovirus (CMV) remain a significant burden in patients undergoing hematopoietic stem cell transplantation (HSCT). The establishment of efficient immunological control, primarily mediated by cytotoxic T cells, plays a critical role in preventing CMV-associated disease in transplant recipients. Recent studies have also begun to investigate the impact genetic variation in CMV has upon disease outcome in transplant recipients. In this study, we sought to investigate the role T cell immunity plays in recognizing and controlling genetic variants of CMV. We demonstrate that while a significant proportion of HSCT recipients may be exposed to multiple genetic variants of CMV, this does not necessarily lead to immune control mediated via recognition of this genetic variation. Rather, immune control is associated with the efficient establishment of a stable immune response predominantly directed against immunodominant conserved T cell epitopes.

Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T-cells that crossreact with selfderived 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 (TCRs) 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, restricted to viruses that infect humans. Peptide sequences were ranked in order of likelihood recognition. For all anti-viral CD8+ T-cell clones examined, 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.

3 Corey Smith, Rebekah M. Brennan, Siok-Keen Tey , Mark J. Smyth , Scott. R. Burrows, 4 John J. Miles, Geoffrey R. Hill 2 and Rajiv Khanna 5 6 QIMR Berghofer Medical Research Institute, QIMR Berghofer Centre for Immunotherapy 7 and Vaccine Development, Brisbane 4029 QLD Australia 8 Bone Marrow Transplant Unit, Royal Brisbane Hospital, Brisbane, 4006 QLD Australia 9 Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, 10 Cardiff CF14 4XN, UK 11 12 Running Title: CMV genetic variation in HSCT recipients 13 14 * Corresponding author 15 Address for correspondence and reprint requests 16 Dr Corey Smith, QIMR Berghofer Medical Research Institute, Tumour Immunology 17 Laboratory, Department of Immunology, 300 Herston Rd, Brisbane, Australia 4006. Tel: 6118 7-3845 3802; Fax: 61-7-38453510; Email: corey.smith@qimrberghofer.edu.au. 19 20 21 22 23 JVI Accepted Manuscript Posted Online 8 June 2016 J. Virol. doi:10.1128/JVI.00297-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1−/− clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1−/− clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.