Jeffrey P. Ward

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Degranulation of Natural Killer Cells Following Interaction with HIV-1-Infected Cells Is Hindered by Downmodulation of NTB-A by Vpu

Natural killer (NK) cell degranulation in response to virus-infected cells is triggered by interactions between invariant NK cell surface receptors and their ligands on target cells. Although HIV-1 Vpr induces expression of ligands for NK cell activation receptor, NKG2D, on infected cells, this is not sufficient to promote lytic granule release. We show that triggering the NK cell coactivation receptor NK-T- and -B cell antigen (NTB-A) alongside NKG2D promotes NK cell degranulation. Normally, NK cell surface NTB-A binds to NTB-A on CD4+ T cells. However, HIV-1 Vpu downmodulates NTB-A on infected T cells. Vpu associates with NTB-A through its transmembrane region without promoting NTB-A degradation. Cells infected with HIV-1 Vpu mutant elicited at least 50% more NK cells to degranulate than wild-type virus. Moreover, NK cells have a higher capacity to lyse HIV-infected cells with a mutant Vpu. Thus, Vpu downmodulation of NTB-A protects the infected cell from lysis by NK cells.

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and –B alleles and against heterologous MHC-Ineg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56neg/CD16pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24pos blasts derived from primary T cells.

HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.

Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

HLA-C and HLA-E reduce antibody-dependent natural killer cell-mediated cytotoxicity of HIV-infected primary T cell blasts.

Objective: To determine whether the presence of HLA-C and HLA-E on HIV-infected cells modulates autologous natural killer (NK) cells from implementing antibody-dependent cell-mediated cytotoxicity (ADCC) of HIV-infected cells. Design: The capability of HLA-C and HLA-E to control NK cell killing of HIV-infected autologous T cells coated with anti-gp120 monoclonal antibody was determined by blocking the interaction between the inhibitory receptors on NK cells and the MHC class I molecules on infected cells. Methods: Phytohemagglutinin-treated CD4 T cells were infected in vitro with HIV-1. Infected cells were separated from uninfected cells by removal of CD4 T cells. Infected cells were labeled with chromium-51, treated with a cocktail of four different monoclonal antibodies against HIV gp120, and co-cultured with freshly isolated autologous NK cells that were incubated with or without anti-CD159a, anti-CD158a, and CD158b, or all three antibodies combined. Killing of the HIV-infected cells by NK cells was assessed in a 4 h cytotoxic assay. Results: When the interaction between NK cell inhibitory receptors (i.e., CD158a, CD158b, and CD159a) and MHC class I molecules (i.e., HLA-C and HLA-E) on HIV-infected autologous T cells was blocked, a drastic increase in killing of anti-gp120-coated HIV-infected cells by NK cells was observed. Conclusion: These studies indicate that the presence of HLA-C and HLA-E molecules on HIV-infected cells may facilitate evasion of NK-mediated killing of antibody-coated HIV-infected cells.

Regulation of CD4 Expression via Recycling by HRES-1/RAB4 Controls Susceptibility to HIV Infection

A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4S27N had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4S27N enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.

The Role of Neoantigens in Naturally Occurring and Therapeutically Induced Immune Responses to Cancer

Definitive experimental evidence from mouse cancer models and strong correlative clinical data gave rise to the Cancer Immunoediting concept that explains the dual host-protective and tumor-promoting actions of immunity on developing cancers. Tumor-specific neoantigens can serve as targets of spontaneously arising adaptive immunity to cancer and thereby determine the ultimate fate of developing tumors. Tumor-specific neoantigens can also function as optimal targets of cancer immunotherapy against established tumors. These antigens are derived from nonsynonymous mutations that occur during cellular transformation and, because they are foreign to the host genome, are not subject to central tolerance. In this review, we summarize the experimental evidence indicating that cancer neoantigens are the source of both spontaneously occurring and therapeutically induced immune responses against cancer. We also review the advances in genomics, bioinformatics, and cancer immunotherapy that have facilitated identification of neoantigens and have moved personalized cancer immunotherapies into clinical trials, with the promise of providing more specific, safer, more effective, and perhaps even more generalizable treatments to cancer patients than current immunotherapies.

Temporally Distinct PD-L1 Expression by Tumor and Host Cells Contributes to Immune Escape

PD-L1 induction on tumor cells is IFNγ-dependent and transient, but becomes IFNγ-independent and long-lived on tumorassociated macrophages. Thus, assessing PD-L1 expression on both tumor and host cells may better stratify patients undergoing PD-1/PD-L1 blockade therapy. Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expressions prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNγ-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNγ dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106–17. ©2017 AACR.

Endogenous Neoantigen-specific CD8 T Cells Identified in Two Glioblastoma Models Using a Cancer Immunogenomics Approach

Immunogenomics were used to identify tumor-specific neoantigens in two well characterized models of glioblastoma. Endogenous immune responses harbored neoantigen-specific T cells within the brain and lymph nodes, providing a tractable system for additional preclinical immunotherapeutic studies in these systems. The “cancer immunogenomics” paradigm has facilitated the search for tumor-specific antigens over the last 4 years by applying comprehensive cancer genomics to tumor antigen discovery. We applied this methodology to identify tumor-specific “neoantigens” in the C57BL/6-derived GL261 and VM/Dk-derived SMA-560 tumor models. Following DNA whole-exome and RNA sequencing, high-affinity candidate neoepitopes were predicted and screened for immunogenicity by ELISPOT and tetramer analyses. GL261 and SMA-560 harbored 4,932 and 2,171 nonsynonymous exome mutations, respectively, of which less than half were expressed. To establish the immunogenicities of H-2Kb and H-2Db candidate neoantigens, we assessed the ability of the epitopes predicted in silico to be the highest affinity binders to activate tumor-infiltrating T cells harvested from GL261 and SMA-560 tumors. Using IFNγ ELISPOT, we confirmed H-2Db–restricted Imp3D81N (GL261) and Odc1Q129L (SMA-560) along with H-2Kb–restricted E2f8K272R (SMA-560) as endogenous tumor-specific neoantigens that are functionally immunogenic. Furthermore, neoantigen-specific T cells to Imp3D81N and Odc1Q129L were detected within intracranial tumors as well as cervical draining lymph nodes by tetramer analysis. By establishing the immunogenicities of predicted high-affinity neoepitopes in these models, we extend the immunogenomics-based neoantigen discovery pipeline to glioblastoma models and provide a tractable system to further study the mechanism of action of T cell–activating immunotherapeutic approaches in preclinical models of glioblastoma. Cancer Immunol Res; 4(12); 1007–15. ©2016 AACR.

Checkpoint blockade immunotherapy reshapes the high-dimensional phenotypic heterogeneity of murine intratumoural neoantigen-specific CD8 + T cells

The analysis of neoantigen-specific CD8+ T cells in tumour-bearing individuals is challenging due to the small pool of tumour antigen-specific T cells. Here we show that mass cytometry with multiplex combinatorial tetramer staining can identify and characterize neoantigen-specific CD8+ T cells in mice bearing T3 methylcholanthrene-induced sarcomas that are susceptible to checkpoint blockade immunotherapy. Among 81 candidate antigens tested, we identify T cells restricted to two known neoantigens simultaneously in tumours, spleens and lymph nodes in tumour-bearing mice. High-dimensional phenotypic profiling reveals that antigen-specific, tumour-infiltrating T cells are highly heterogeneous. We further show that neoantigen-specific T cells display a different phenotypic profile in mice treated with anti-CTLA-4 or anti-PD-1 immunotherapy, whereas their peripheral counterparts are not affected by the treatments. Our results provide insights into the nature of neoantigen-specific T cells and the effects of checkpoint blockade immunotherapy.Immune checkpoint blockade (ICB) therapies can unleash anti-tumour T-cell responses. Here the authors show, by integrating MHC tetramer multiplexing, mass cytometry and high-dimensional analyses, that neoantigen-specific, tumour-infiltrating T cells are highly heterogeneous and are subjected to ICB modulations.