Upregulated Glucose Metabolism Correlates Inversely with CD8(+) T-cell Infiltration and Survival in Squamous Cell Carcinoma

Antibodies that block T-cell–regulatory checkpoints have recently emerged as a transformative approach to cancer treatment. However, the clinical efficacy of checkpoint blockade depends upon inherent tumor immunogenicity, with variation in infiltrating T cells contributing to differences in objective response rates. Here, we sought to understand the molecular correlates of tumor-infiltrating T lymphocytes (TIL) in squamous cell carcinoma (SCC), using a systems biologic approach to integrate publicly available omics datasets with histopathologic features. We provide evidence that links TIL abundance and therapeutic outcome to the regulation of tumor glycolysis by EGFR and HIF, both of which are attractive molecular targets for use in combination with immunotherapeutics

Human Papillomavirus Drives Tumor Development Throughout the Head and Neck: Improved Prognosis Is Associated With an Immune Response Largely Restricted to the Oropharynx

Purpose In squamous cell carcinomas of the head and neck (HNSCC), the increasing incidence of oropharyngeal squamous cell carcinomas (OPSCCs) is attributable to human papillomavirus (HPV) infection. Despite commonly presenting at late stage, HPV-driven OPSCCs are associated with improved prognosis compared with HPV-negative disease. HPV DNA is also detectable in nonoropharyngeal (non-OPSCC), but its pathogenic role and clinical significance are unclear. The objectives of this study were to determine whether HPV plays a causal role in non-OPSCC and to investigate whether HPV confers a survival benefit in these tumors. Methods Meta-analysis was used to build a cross-tissue gene-expression signature for HPV-driven cancer. Classifiers trained by machine-learning approaches were used to predict the HPV status of 520 HNSCCs profiled by The Cancer Genome Atlas project. DNA methylation data were similarly used to classify 464 HNSCCs and these analyses were integrated with genomic, histopathology, and survival data to permit a comprehensive comparison of HPV transcript-positive OPSCC and non-OPSCC. Results HPV-driven tumors accounted for 4.1% of non-OPSCCs. Regardless of anatomic site, HPV+ HNSCCs shared highly similar gene expression and DNA methylation profiles; nonkeratinizing, basaloid histopathological features; and lack of TP53 or CDKN2A alterations. Improved overall survival, however, was largely restricted to HPV-driven OPSCCs, which were associated with increased levels of tumor-infiltrating lymphocytes compared with HPV-driven non-OPSCCs. Conclusion Our analysis identified a causal role for HPV in transcript-positive non-OPSCCs throughout the head and neck. Notably, however, HPV-driven non-OPSCCs display a distinct immune microenvironment and clinical behavior compared with HPV-driven OPSCCs

Fc-γ receptor-mediated crosslinking co-defines the immunostimulatory activity of anti-human CD96 antibodies.

New strategies that augment T-cell responses are required to broaden the therapeutic arsenal against cancer. CD96, TIGIT and CD226 are receptors that bind to a communal ligand, CD155, and transduce either inhibitory or activating signals. Whereas the function of TIGIT and CD226 is established, the role of CD96 remains ambiguous. Using a panel of engineered antibodies, we discovered that the T-cell stimulatory activity of anti-CD96 antibodies requires antibody crosslinking and is potentiated by Fc-gamma receptors. Thus, soluble 'Fc silent' anti-CD96 antibodies failed to stimulate human T cells, whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably, the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and was dependent on antibody trans-crosslinking by Fc-γRI. In contrast, neither human IgG2 nor variants with increased Fc-γ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T-cell activation, leading to proliferation, cytokine secretion and resistance to regulatory T-cell suppression. Furthermore, CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its crosslinking activated tumor-infiltrating T cells, thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy. 

A subset of myofibroblastic cancer-associated fibroblasts regulate collagen fiber elongation, which is prognostic in multiple cancers

Collagen structure has been shown to influence tumor cell invasion, metastasis and clinical outcome in breast cancer. However, it remains unclear how it affects other solid cancers. Here we utilized multi-photon laser scanning microscopy and Second Harmonic Generation to identify alterations to collagen fiber structure within the tumor stroma of head & neck, esophageal and colorectal cancers. Image segmentation algorithms were then applied to quantitatively characterize these morphological changes, showing that elongated collagen fibers significantly correlated with poor clinical outcome (Log Rank p < 0.05). We used TGF-? treatment to model fibroblast conversion to smooth muscle actin SMA-positive cancer associated fibroblasts (CAFs) and found that these cells induce the formation of elongated collagen fibers in vivo. However, proteomic/transcriptomic analysis of SMA-positive CAFs cultured ex-vivo showed significant heterogeneity in the expression of genes with collagen fibril organizing gene ontology. Notably, stratifying patients according to stromal SMA-positivity and collagen fiber elongation was found to provide a highly significant correlation with poor survival in all 3 cancer types (Log Rank p ? 0.003). In summary, we show that increased collagen fiber length correlates with poor patient survival in multiple tumor types and that only a sub-set of SMA-positive CAFs can mediate the formation of this collagen structure

LILRB3 (ILT5) is a myeloid cell checkpoint that elicits profound immunomodulation.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor

Therapeutic gene transfer for malignant melanoma : augmentation of direct versus indirect tumour antigen presentation

The worldwide incidence of cutaneous malignant melanoma has risen markedly during the last two decades, and by the year 2000, it is estimated that 1 in 200 individuals in the U.K. may develop metastatic melanoma during their lifetime. Critically, the current therapeutic regimes for the treatment of metastatic melanoma have largely failed to have a significant impact on long-term-survival, prompting a search for an effective adjuvant therapy for patients with disseminated disease following surgery. The summation of these findings has culminated in the emergence of therapeutic gene transfer as a potential means of eradicating residual disease.;This study sought to compare the genetic modification of melanoma cells such that they can directly present tumour-associated antigens to T-cells, with strategies designed to promote tumour antigen-presentation by dendritic cells (DC). Specifically, the human melanoma cell line A-375 was transfected to express CD80 and assessed for its capacity to activate naive T-cells and to prime specific cytotoxic T-lymphocytes (CTL). In addition, A-375 cells, transfected to express granulocyte-macrophage colony-stimulating factor and interleukin-4, were examined for their capacity to support the functional maturation of DC from the peripheral blood, with a view to performing in vitro CTL priming experiments.;Rather than clarifying which strategy offered the greater potential for augmenting anti-melanoma CTL responses, the results described add further to the current uncertainties regarding the treatment of cancer by gene transfer and stress the need for caution when considering the appropriate gene(s) for transfection. They also highlight the role of melanoma-derived cytokines in mediating immunosuppression

Prolonged antigen expression following DNA vaccination impairs effector CD8(+) T cell function and memory development

After priming, naive T cells undergo a program of expansion, contraction, and memory formation. Numerous studies have indicated that only a brief period of antigenic stimulation is required to fully commit CD8(+) T cells to this program. Nonetheless, the persistence of Ag may modulate the eventual fate of CD8(+) T cells. Using DNA delivery, we showed previously that direct presentation primes high levels of effector CD8(+) T cells as compared with cross-presentation. One explanation now revealed is that prolonged cross-presentation limits effector cell expansion and function. To analyze this, we used a drug-responsive system to regulate Ag expression after DNA injection. Reducing expression to a single burst expanded greater numbers of peptide-specific effector CD8(+) T cells than sustained Ag. Consequences for memory development were assessed after boosting and showed that, although persistent Ag maintained higher numbers of tetramer-positive CD8(+) T cells, these expanded less (similar to 4-fold) than those induced by transient Ag expression (similar to 35-fold). Transient expression at priming therefore led to a net higher secondary response. In terms of vaccine design, we propose that the most effective DNA-based CD8(+) T cell vaccines will be those that deliver a short burst of Ag

Vaccination with DNA encoding a single-chain TCR fusion protein induces anticlonotypic immunity and protects against T-cell lymphoma

The clonotypic T-cell antigen receptor (TCR) provides unique V? and Vßsequences with potential as idiotypic targets for immunoregulation. For T-cell malignancies, vaccination with the TCR could induce therapeutic anti-idiotypic responses. To facilitate this approach, we have developed DNA vaccines that include the genes encoding TCR sequences from a T-cell lymphoma (TCL). To combine requirements for stable folding with a simple minimized single-chain construction, we used a three-domain V?VßCß sequence. To promote anti-TCR immunity, we fused a pathogen-derived sequence from tetanus toxin to the 3'-end of the single-chain TCR. The fusion gene vaccine induced anti-idiotypic antibodies and generated protection against the TCL. The critical requirement for the conformational integrity of the delivered TCR antigen was highlighted by the observation that DNA fusion vaccines containing either V?Vß or VßCß sequences failed to generate antibodies reactive with the native TCR or provide protection. This is the first report of a DNA vaccine able to induce anti-idiotypic immunity against TCL, and it presents a simple strategy for selectively eliminating T-cell clones in vivo

Prime-boost with alternating DNA vaccines designed to engage different antigen presentation pathways generates high frequencies of peptide-specific CD8+ T cells

The route for presentation of Ag to CD8+ or CD4+ T cells following DNA vaccination is critical for determining outcome, but the pathways involved are unclear. In this study, we compare two different DNA vaccine designs aimed to elicit CD8+ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4+ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of Th responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated Th-dependent priming of CD8+ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8+ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization