Mechanism of EBV inducing anti-tumour immunity and its therapeutic use.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein-Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein-Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

Corrigendum: An immunogenic personal neoantigen vaccine for patients with melanoma.

This corrects the article DOI: 10.1038/nature22991.

An immunogenic personal neoantigen vaccine for patients with melanoma.

Effective anti-tumour immunity in humans has been associated with the presence of T cells directed at cancer neoantigens, a class of HLA-bound peptides that arise from tumour-specific mutations. They are highly immunogenic because they are not present in normal tissues and hence bypass central thymic tolerance. Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their systematic discovery and evaluation only became feasible with the recent availability of massively parallel sequencing for detection of all coding mutations within tumours, and of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules. We hypothesized that vaccination with neoantigens can both expand pre-existing neoantigen-specific T-cell populations and induce a broader repertoire of new T-cell specificities in cancer patients, tipping the intra-tumoural balance in favour of enhanced tumour control. Here we demonstrate the feasibility, safety, and immunogenicity of a vaccine that targets up to 20 predicted personal tumour neoantigens. Vaccine-induced polyfunctional CD4+ and CD8+ T cells targeted 58 (60%) and 15 (16%) of the 97 unique neoantigens used across patients, respectively. These T cells discriminated mutated from wild-type antigens, and in some cases directly recognized autologous tumour. Of six vaccinated patients, four had no recurrence at 25 months after vaccination, while two with recurrent disease were subsequently treated with anti-PD-1 (anti-programmed cell death-1) therapy and experienced complete tumour regression, with expansion of the repertoire of neoantigen-specific T cells. These data provide a strong rationale for further development of this approach, alone and in combination with checkpoint blockade or other immunotherapies.

A cloning and expression system to probe T-cell receptor specificity and assess functional avidity to neoantigens.

Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRß, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system's capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut-MGA, and use this approach to identify the mut-MGA-specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies.

Functional heterogeneity in the CD4+ T cell response to murine γ-herpesvirus 68.

CD4(+) T cells are critical for the control of virus infections, T cell memory, and immune surveillance. We studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4(+) T cells using gp150-specific TCR-transgenic mice. This allowed a more detailed study of the characteristics of the CD4(+) T cell response than did previously available approaches for this virus. Most gp150-specific CD4(+) T cells expressed T-bet and produced IFN-γ, indicating that MHV-68 infection triggered differentiation of CD4(+) T cells largely into the Th1 subset, whereas some became follicular Th cells and Foxp3(+) regulatory T cells. These CD4(+) T cells were protective against MHV-68 infection in the absence of CD8(+) T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4(+) T cells, based on lymphocyte Ag 6C (Ly6C) expression. Ly6C expression positively correlated with IFN-γ, TNF-α, and granzyme B production; T-bet and KLRG1 expression; proliferation; and CD4(+) T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression, and secondary expansion potential. Ly6C(+) and Ly6C(-) gp150-specific CD4(+) T cells were able to interconvert in a bidirectional manner upon secondary Ag exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4(+) T cells but is inversely correlated with memory potential. Interconversion between Ly6C(+) and Ly6C(-) cells may maintain a balance between the two Ag-specific CD4(+) T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4(+) T cells during persistent virus infection.

MCL1 enhances the survival of CD8+ memory T Cells after viral infection.

UNLABELLED: Viral infection results in the generation of massive numbers of activated effector CD8(+) T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon reexposure to antigen even at protracted times postinfection. Here, antiapoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing in the proportion of CD8(+) T cells, away from SLECs toward MPECs, during the acute phase of vaccinia virus infection. A higher frequency and total number of antigen-specific CD8(+) T cells were observed in MCL1 transgenic mice. These findings show that MCL1 can shape the makeup of the CD8(+) T cell response, promoting the formation of long-term memory. IMPORTANCE: During an immune response to a virus, CD8(+) T cells kill cells infected by the virus, and most die when the infection resolves. However, a small proportion of cells survives and differentiates into long-lived memory cells that confer protection from reinfection by the same virus. This report shows that transgenic expression of an MCL1 protein enhances survival of memory CD8(+) T cells following infection with vaccinia virus. This is important because it shows that MCL1 expression may be an important determinant of the formation of long-term CD8(+) T cell memory.

Immune escape of γ-herpesviruses from adaptive immunity.

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are two γ-herpesviruses identified in humans and are strongly associated with the development of malignancies. Murine γ-herpesvirus (MHV-68) is a naturally occurring rodent pathogen, representing a unique experimental model for dissecting γ-herpesvirus infection and the immune response. These γ-herpesviruses actively antagonize the innate and adaptive antiviral responses, thereby efficiently establishing latent or persistent infections and even promoting development of malignancies. In this review, we summarize immune evasion strategies of γ-herpesviruses. These include suppression of MHC-I-restricted and MHC-II-restricted antigen presentation, impairment of dendritic cell functions, downregulation of costimulatory molecules, activation of virus-specific regulatory T cells, and induction of inhibitory cytokines. There is a focus on how both γ-herpesvirus-derived and host-derived immunomodulators interfere with adaptive antiviral immunity. Understanding immune-evasive mechanisms is essential for developing future immunotherapies against EBV-driven and KSHV-driven tumors.

Regulatory CD8+ T cells associated with erosion of immune surveillance in persistent virus infection suppress in vitro and have a reversible proliferative defect.

CD4(+) T cell help is critical for CD8(+) T cell memory and immune surveillance against persistent virus infections. Our recent data have showed the lack of CD4(+) T cells leads to the generation of an IL-10-producing CD8(+) T cell population during persistent murine γ-herpesvirus-68 (MHV-68) infection. IL-10 from these cells is partly responsible for erosion in immune surveillance, leading to spontaneous virus reactivation in lungs. In this study, we further characterized the generation, phenotype, and function of these IL-10-producing CD8(+) T cells by comparing with a newly identified IL-10-producing CD8(+) T cell population present during the acute stage of the infection. The IL-10-producing CD8(+) populations in acute and chronic stages differed in their requirement for CD4(+) T cell help, the dependence on IL-2/CD25 and CD40-CD40L pathways, and the ability to proliferate in vitro in response to anti-CD3 stimulation. IL-10-producing CD8(+) T cells in the chronic stage showed a distinct immunophenotypic profile, sharing partial overlap with the markers of previously reported regulatory CD8(+) T cells, and suppressed the proliferation of naive CD8(+) T cells. Notably, they retained the ability to produce effector cytokines and cytotoxic activity. In addition, the proliferative defect of the cells could be restored by addition of exogenous IL-2 or blockade of IL-10. These data suggest that the IL-10-producing CD8(+) T cells arising in chronic MHV-68 infection in the absence of CD4(+) T cell help belong to a subset of CD8(+) regulatory T cells.

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy.

High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.

Identification of plasmid- and integron-borne blaIMP-1 and blaIMP-10 in clinical isolates of Serratia marcescens.

The emergence of carbapenem-hydrolysing metallo-beta-lactamases (MBLs) is a serious threat to the clinical utility of carbapenems. This study identified plasmid- and integron-borne bla(IMP-1) and bla(IMP-10) in clinical isolates of Serratia marcescens. The bla(IMP-1) and bla(IMP-10) gene cassettes were carried by a class 1 integron and followed by the aac(6')-IIc gene cassette. The bla(IMP-1) and bla(IMP-10) gene cassettes were preceded by a weak P(ant) promoter, TGGACA(N)(17)TAAGCT, and an inactive P2 promoter, TTGTTA(N)(14)TACAGT. These genes were easily transferred to Escherichia coli by conjugation and transformation, indicating that they are located on transferable plasmids. Due to the acquisition of bla(IMP-1), the susceptibility of E. coli transconjugants to imipenem, meropenem, panipenem and biapenem decreased by 32-, 256-, 64- and 128-fold, respectively. In comparison, after gaining bla(IMP-10), the susceptibility of E. coli transconjugants to the four carbapenems decreased by 64-, 2048-, 256- and 64-fold, respectively. Strains harbouring bla(IMP-10) showed higher-level resistance to imipenem, meropenem and panipenem than the strains harbouring bla(IMP-1), although the nucleotide sequences of the class 1 integrons carrying bla(IMP-10) and bla(IMP-1) were identical except for a single point mutation.

Contributions of IMP-10 metallo-beta-lactamase, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa.

BACKGROUND: The emergence of carbapenem-hydrolyzing metallo-beta-lactamases (MBLs) is a serious threat to clinical medication of carbapenems. We evaluated the contributions of IMP-10 MBL, the outer membrane barrier and the MexAB-OprM efflux system to high-level carbapenem resistance in Pseudomonas aeruginosa clinical isolates. METHODS: MBL-producing strains were screened by ceftazidime and mercaptoacetic acid double-disk synergy testing. Genes were determined by PCR analysis and DNA sequencing. IMP-10 MBL activity was assayed using nitrocefin as a substrate. RESULTS: The bla(IMP-10)+ strains showed high-level resistance to carbapenems, with minimum inhibitory concentrations of 512 to >4,096 microg/ml. The minimum inhibitory concentrations were decreased by the inhibitors of MBLs, sodium mercaptoacetate (SMA) and ethylenediaminetetraacetic acid (EDTA), 16 and 64 fold, respectively. However, against the activity of the prepared IMP-10 MBL, the 50% inhibitory concentrations of SMA and EDTA were 5.5 and 211.6 microM, respectively, indicating that SMA was much more effective than EDTA in blocking the enzyme activity. The contradictory results may be explained by the additional effect of EDTA as an outer membrane permeabilizer because the susceptibility of the bla(IMP)- strains to carbapenems was increased 2 fold by EDTA, but not by SMA. In the presence of carbonyl cyanide M-chlorophenylhydrazone, a proton motive force inhibitor, the susceptibility of the P. aeruginosa isolates to carbapenems was increased 1-4 fold. CONCLUSION: The acquired IMP-10 MBL is a crucial factor in the high-level resistance to carbapenems in P. aeruginosa clinical isolates, while the outer membrane barrier and the MexAB-OprM efflux system play a basic and minor role, respectively.

Towards personalized, tumour-specific, therapeutic vaccines for cancer.

Cancer vaccines, which are designed to amplify tumour-specific T cell responses through active immunization, have long been envisioned as a key tool of effective cancer immunotherapy. Despite a clear rationale for such vaccines, extensive past efforts were unsuccessful in mediating clinically relevant antitumour activity in humans. Recently, however, next-generation sequencing and novel bioinformatics tools have enabled the systematic discovery of tumour neoantigens, which are highly desirable immunogens because they arise from somatic mutations of the tumour and are therefore tumour specific. As a result of the diversity of tumour neoepitopes between individuals, the development of personalized cancer vaccines is warranted. Here, we review the emerging field of personalized cancer vaccination and discuss recent developments and future directions for this promising treatment strategy.

CD4(+) T-cell dependence of primary CD8(+) T-cell response against vaccinia virus depends upon route of infection and viral dose.

CD4(+) T-cell help (CD4 help) plays a pivotal role in CD8(+) T-cell responses against viral infections. However, the role in primary CD8(+) T-cell responses remains controversial. We evaluated the effects of infection route and viral dose on primary CD8(+) T-cell responses to vaccinia virus (VACV) in MHC class II(-/-) mice. CD4 help deficiency diminished the generation of VACV-specific CD8(+) T cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. A large viral dose could not restore normal expansion of VACV-specific CD8(+) T cells in i.p. infected MHC II(-/-) mice. In contrast, dependence on CD4 help was observed in i.n. infected MHC II(-/-) mice when a small viral dose was used. These data suggested that primary CD8(+) T-cell responses are less dependent on CD4 help in i.n. infection compared to i.p. infection. Activated CD8(+) T cells produced more IFN-γ, TNF-α and granzyme B in i.n. infected mice than those in i.p. infected mice, regardless of CD4 help. IL-2 signaling via CD25 was not necessary to drive expansion of VACV-specific CD8(+) T cells in i.n. infection, but it was crucial in i.p. infection. VACV-specific CD8(+) T cells underwent increased apoptosis in the absence of CD4 help, but proliferated normally and had cytotoxic potential, regardless of infection route. Our results indicate that route of infection and viral dose are two determinants for CD4 help dependence, and intranasal infection induces more potent effector CD8(+) T cells than i.p. infection.

Strain-dependent requirement for IFN-γ for respiratory control and immunotherapy in murine gammaherpesvirus infection.

Interferon-γ (IFN-γ) and perforin (pfp) are important effector mechanisms used by CD8 T cells to clear virus-infected cells. In this study, we used IFN-γ/pfp double knockout mice to address if these two effector molecules play redundant roles in the control of acute infection with murine gammaherpesvirus-68 (MHV-68) in BALB/C mice. Perforin knockout (KO) mice and wild-type mice cleared infectious virus from the lungs, even following high-dose infection. However, the IFN-γ KO and IFN-γ/pfp double knockout (DKO) groups had higher virus titers in the lungs at day 10 post-infection, and both groups had higher mortality rates. In IFN-γ/pfp DKO mice, the virus titer and mortality rate were significant higher than in IFN-γ KO mice, indicating a role for perforin in protection from disease. WT mice given IFN-γ blocking antibody also showed significantly higher viral titers. In contrast, IFN-γ KO mice on a C57BL/6 background controlled respiratory infection comparably to wild-type mice. These data show that perforin plays a redundant role in the control of virus replication, but IFN-γ plays an essential role in BALB/C mice infected with MHV-68. We conclude that there is a marked strain-dependent difference in the effector mechanisms needed to control acute MHV-68 infection between C57BL/6 and BALB/C mice. In addition we show that immune therapy that re-establishes viral control after spontaneous reactivation in CD4-deficient mice depends upon perforin in C57BL/6 mice but IFN-γ in BALB/C mice.