Reprogramming tumor-associated macrophages to outcompete endovascular endothelial progenitor cells and suppress tumor neoangiogenesis.

Tumors develop by invoking a supportive environment characterized by aberrant angiogenesis and infiltration of tumor-associated macrophages (TAMs). In a transgenic model of breast cancer, we found that TAMs localized to the tumor parenchyma and were smaller than mammary tissue macrophages. TAMs had low activity of the metabolic regulator mammalian/mechanistic target of rapamycin complex 1 (mTORC1), and depletion of negative regulator of mTORC1 signaling, tuberous sclerosis complex 1 (TSC1), in TAMs inhibited tumor growth in a manner independent of adaptive lymphocytes. Whereas wild-type TAMs exhibited inflammatory and angiogenic gene expression profiles, TSC1-deficient TAMs had a pro-resolving phenotype. TSC1-deficient TAMs relocated to a perivascular niche, depleted protein C receptor (PROCR)-expressing endovascular endothelial progenitor cells, and rectified the hyperpermeable blood vasculature, causing tumor tissue hypoxia and cancer cell death. TSC1-deficient TAMs were metabolically active and effectively eliminated PROCR-expressing endothelial cells in cell competition experiments. Thus, TAMs exhibit a TSC1-dependent mTORC1-low state, and increasing mTORC1 signaling promotes a pro-resolving state that suppresses tumor growth, defining an innate immune tumor suppression pathway that may be exploited for cancer immunotherapy.

Tumor-associated macrophages expressing the transcription factor IRF8 promote T cell exhaustion in cancer.

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.

Differences in Tumor-Associated T cell receptor repertoires between Early-Onset and Average-Onset colorectal cancer.

The incidence of colorectal cancer (CRC) among individuals younger than age 50 (early onset CRC; EOCRC) has substantially increased, yet the etiology and molecular mechanisms underlying this alarming rise remain unclear. We compared tumor-associated T cell repertoires between EOCRC and average-onset CRC (AOCRC) to uncover potentially unique immune microenvironment-related features by age of onset. Our discovery cohort included 242 patients who underwent surgical resection at Cleveland Clinic from 2000 to 2020. EOCRC was defined as age < 50 years at diagnosis (N = 126), and AOCRC as age ≥ 60 years (N = 116). T cell receptor (TCR) abundance and clonality were measured by immunosequencing of tumors. Logistic regression models were used to evaluate the associations between TCR repertoire features and age of onset, adjusting for sex, race, tumor location, and stage. Findings were replicated in 152 EOCRC and 1,984 AOCRC cases from the Molecular Epidemiology of Colorectal Cancer Study. EOCRC tumors had significantly higher TCR diversity compared to AOCRC tumors in the discovery cohort (Odds Ratio (OR):0.44, 95% Confidence Interval (CI):0.32-0.61, p < .0001). This association was also observed in the replication cohort (OR : 0.74, 95% CI : 0.62-0.89, p = .0013). No significant differences in TCR abundance were observed between EOCRC and AOCRC in either cohort. Higher TCR diversity, suggesting a more diverse intratumoral T cell response, is more frequently observed in EOCRC than AOCRC. Further studies are warranted to investigate the role of T cell diversity and the adaptive immune response more broadly in the etiology and outcomes of EOCRC.

Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide.

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.

Nivolumab plus ipilimumab in advanced salivary gland cancer: a phase 2 trial.

Salivary gland cancers (SGCs) are rare, aggressive cancers without effective treatments when metastasized. We conducted a phase 2 trial evaluating nivolumab (nivo, anti-PD-1) and ipilimumab (ipi, anti-CTLA-4) in 64 patients with metastatic SGC enrolled in two histology-based cohorts (32 patients each): adenoid cystic carcinoma (ACC; cohort 1) and other SGCs (cohort 2). The primary efficacy endpoint (≥4 objective responses) was met in cohort 2 (5/32, 16%) but not in cohort 1 (2/32, 6%). Treatment safety/tolerability and progression-free survival (PFS) were secondary endpoints. Treatment-related adverse events grade ≥3 occurred in 24 of 64 (38%) patients across both cohorts, and median PFS was 4.4 months (95% confidence interval (CI): 2.4, 8.3) and 2.2 months (95% CI: 1.8, 5.3) for cohorts 1 and 2, respectively. We present whole-exome, RNA and T cell receptor (TCR) sequencing data from pre-treatment and on-treatment tumors and immune cell flow cytometry and TCR sequencing from peripheral blood at serial timepoints. Responding tumors universally demonstrated clonal expansion of pre-existing T cells and mutational contraction. Responding ACCs harbored neoantigens, including fusion-derived neoepitopes, that induced T cell responses ex vivo. This study shows that nivo+ipi has limited efficacy in ACC, albeit with infrequent, exceptional responses, and that it could be promising for non-ACC SGCs, particularly salivary duct carcinomas. ClinicalTrials.gov identifier: NCT03172624 .

Functional landscapes of POLE and POLD1 mutations in checkpoint blockade-dependent antitumor immunity.

Defects in pathways governing genomic fidelity have been linked to improved response to immune checkpoint blockade therapy (ICB). Pathogenic POLE/POLD1 mutations can cause hypermutation, yet how diverse mutations in POLE/POLD1 influence antitumor immunity following ICB is unclear. Here, we comprehensively determined the effect of POLE/POLD1 mutations in ICB and elucidated the mechanistic impact of these mutations on tumor immunity. Murine syngeneic tumors harboring Pole/Pold1 functional mutations displayed enhanced antitumor immunity and were sensitive to ICB. Patients with POLE/POLD1 mutated tumors harboring telltale mutational signatures respond better to ICB than patients harboring wild-type or signature-negative tumors. A mutant POLE/D1 function-associated signature-based model outperformed several traditional approaches for identifying POLE/POLD1 mutated patients that benefit from ICB. Strikingly, the spectrum of mutational signatures correlates with the biochemical features of neoantigens. Alterations that cause POLE/POLD1 function-associated signatures generate T cell receptor (TCR)-contact residues with increased hydrophobicity, potentially facilitating T cell recognition. Altogether, the functional landscapes of POLE/POLD1 mutations shape immunotherapy efficacy.

Single-cell sequencing links multiregional immune landscapes and tissue-resident T cells in ccRCC to tumor topology and therapy efficacy.

Clear cell renal cell carcinomas (ccRCCs) are highly immune infiltrated, but the effect of immune heterogeneity on clinical outcome in ccRCC has not been fully characterized. Here we perform paired single-cell RNA (scRNA) and T cell receptor (TCR) sequencing of 167,283 cells from multiple tumor regions, lymph node, normal kidney, and peripheral blood of two immune checkpoint blockade (ICB)-naïve and four ICB-treated patients to map the ccRCC immune landscape. We detect extensive heterogeneity within and between patients, with enrichment of CD8A+ tissue-resident T cells in a patient responsive to ICB and tumor-associated macrophages (TAMs) in a resistant patient. A TCR trajectory framework suggests distinct T cell differentiation pathways between patients responding and resistant to ICB. Finally, scRNA-derived signatures of tissue-resident T cells and TAMs are associated with response to ICB and targeted therapies across multiple independent cohorts. Our study establishes a multimodal interrogation of the cellular programs underlying therapeutic efficacy in ccRCC.

Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

Cell-selective lysis by novel analogues of melittin against human red blood cells and Escherichia coli.

Melittin is a good model antimicrobial peptide to understand the basis of its lytic activities against bacteria and mammalian cells. Novel analogues of melittin were designed by substituting the leucine residue(s) at the "d" and "a" positions of its previously identified leucine zipper motif. A scrambled peptide having the same composition of melittin with altered leucine zipper sequence was also designed. The analogues of melittin including the scrambled peptide showed a drastic reduction in cytotoxicity though they exhibited comparable bactericidal activities. Only melittin but not its analogues localized strongly onto hRBCs and formed pores of approximately 2.2-3.4 nm. However, melittin and its analogues localized similarly onto Escherichia coli and formed pores of varying sizes as tested onto Bacillus megaterium. The data showed that the substitution of hydrophobic leucine residue(s) by lesser hydrophobic alanine residue(s) in the leucine zipper sequence of melittin disturbed its pore-forming activity and mechanism only in hRBCs but not in the tested bacteria.

Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences.

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple 'a' position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.

Diverse Neoantigens and the Development of Cancer Therapies.

Cancer is the manifestation of uncontrolled cellular growth and immune escape mechanisms. Unrestrained tumor growth can be associated with incidental errors in the genome during replication and genotoxic agents can alter the structure and sequence of our DNA. Among all genetic aberrations in cancer, only limited number of mutations can produce immunogenic antigens which have the potential to bind human leukocyte antigen class I or human leukocyte antigen class II, and help activate the adaptive immune system. These neoantigens can be recognized by CD8+ and CD4+ neoantigen-specific T lymphocytes. Recently, several immune checkpoint targeting drugs have been approved for clinical use. Primarily, these drugs expand and facilitate the cytotoxic activity of neoantigen-specific T cells to eradicate tumors. Differential drug response across cancers could be attributed, at least in part, to differences in the 'tumor antigen landscape' and 'antigen presentation pathway' in patients. Although tumor mutational burden correlates with response to immune checkpoint inhibitors in many cancer types and has evolved as a broad biomarker, a comprehensive understanding of the neoantigen landscape and the function of cognate T cell responses is lacking and is needed for improved patient selection criteria and neoantigen vaccine design. Here, we review cancer neoantigens, their implications for antitumor responses, the dynamics of neoantigen-specific T cells, and the advancement of neoantigen-based therapy in proposed clinical trials.

Commensal bacteria stimulate antitumor responses via T cell cross-reactivity.

Recent studies show gut microbiota modulate antitumor immune responses; one proposed mechanism is cross-reactivity between antigens expressed in commensal bacteria and neoepitopes. We found that T cells targeting an epitope called SVYRYYGL (SVY), expressed in the commensal bacterium Bifidobacterium breve (B. breve), cross-react with a model neoantigen, SIYRYYGL (SIY). Mice lacking B. breve had decreased SVY-reactive T cells compared with B. breve-colonized mice, and the T cell response was transferable by SVY immunization or by cohousing mice without Bifidobacterium with ones colonized with Bifidobacterium. Tumors expressing the model SIY neoantigen also grew faster in mice lacking B. breve compared with Bifidobacterium-colonized animals. B. breve colonization also shaped the SVY-reactive TCR repertoire. Finally, SVY-specific T cells recognized SIY-expressing melanomas in vivo and led to decreased tumor growth and extended survival. Our work demonstrates that commensal bacteria can stimulate antitumor immune responses via cross-reactivity and how bacterial antigens affect the T cell landscape.

Design of nontoxic analogues of cathelicidin-derived bovine antimicrobial peptide BMAP-27: the role of leucine as well as phenylalanine zipper sequences in determining its toxicity.

BMAP-27 is a cathelicidin-derived bovine antimicrobial peptide, which shows moderate cytotoxicity and potent antibacterial activity against a wide variety of microorganisms. Despite a number of studies, very little is known about the amino acid sequences of this peptide that controls its antibacterial and cytotoxic activities. Small stretches of phenylalanine and leucine zipper sequences were identified at the N- and C-termini of the molecule, respectively. To understand the structural and functional roles of these sequence elements, we synthesized and characterized several analogues of BMAP-27 after substituting leucine or phenylalanine residue(s) at a and/or d positions of the leucine and phenylalanine zipper sequences, respectively, with alanine. BMAP-27 analogues exhibited significantly reduced cytotoxicity against the human red blood (hRBC) and murine 3T3 cells as compared to that of the wild-type peptide. Interestingly, BMAP-27 and its analogues exhibited comparable antibacterial activity against the selected Gram-positive and Gram-negative bacteria. Moreover, BMAP-27 and its analogues exhibited similar localization and assembly onto the selected bacteria and induced comparable permeability in these cells. However, only BMAP-27, not its analogues, assembled and bound strongly onto the hRBCs and permeabilized them. The results indicated that not only a leucine zipper but also a phenylalanine zipper sequence plays an important role in maintaining the assembly of BMAP-27 on the mammalian cells examined here and cytotoxic activity against them. To the best of our knowledge, this is the first report of the evaluation of structural and functional roles of a phenylalanine zipper sequence in a naturally occurring antimicrobial peptide.

Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response.

Tumors with mismatch repair deficiency (MMR-d) are characterized by sequence alterations in microsatellites and can accumulate thousands of mutations. This high mutational burden renders tumors immunogenic and sensitive to programmed cell death-1 (PD-1) immune checkpoint inhibitors. Yet, despite their tumor immunogenicity, patients with MMR-deficient tumors experience highly variable responses, and roughly half are refractory to treatment. We present experimental and clinical evidence showing that the degree of microsatellite instability (MSI) and resultant mutational load, in part, underlies the variable response to PD-1 blockade immunotherapy in MMR-d human and mouse tumors. The extent of response is particularly associated with the accumulation of insertion-deletion (indel) mutational load. This study provides a rationale for the genome-wide characterization of MSI intensity and mutational load to better profile responses to anti-PD-1 immunotherapy across MMR-deficient human cancers.

Immunogenic neoantigens derived from gene fusions stimulate T cell responses.

Anti-tumor immunity is driven by self versus non-self discrimination. Many immunotherapeutic approaches to cancer have taken advantage of tumor neoantigens derived from somatic mutations. Here, we demonstrate that gene fusions are a source of immunogenic neoantigens that can mediate responses to immunotherapy. We identified an exceptional responder with metastatic head and neck cancer who experienced a complete response to immune checkpoint inhibitor therapy, despite a low mutational load and minimal pre-treatment immune infiltration in the tumor. Using whole-genome sequencing and RNA sequencing, we identified a novel gene fusion and demonstrated that it produces a neoantigen that can specifically elicit a host cytotoxic T cell response. In a cohort of head and neck tumors with low mutation burden, minimal immune infiltration and prevalent gene fusions, we also identified gene fusion-derived neoantigens that generate cytotoxic T cell responses. Finally, analyzing additional datasets of fusion-positive cancers, including checkpoint-inhibitor-treated tumors, we found evidence of immune surveillance resulting in negative selective pressure against gene fusion-derived neoantigens. These findings highlight an important class of tumor-specific antigens and have implications for targeting gene fusion events in cancers that would otherwise be less poised for response to immunotherapy, including cancers with low mutational load and minimal immune infiltration.

Immunomodulatory effects of curcumin: in-vivo.

Curcumin specifically exhibits cytostatic and cytotoxic effects against tumors of multiple origin. Previously we have demonstrated apoptotic activity of curcumin against tumor cells with no effect on normal cells in-vitro. Many anti-cancer drugs exhibit deleterious effects on immune cells, which restrict their wide use in-vivo. In the present study, we have evaluated the effect of curcumin on the major functions of T cells, natural killer cells, macrophages and on total splenocytes in-vivo, which insight the role of curcumin on their broad effector functions. This study demonstrates that prolonged curcumin-injections (i.p.) do not impair the cytotoxic function of natural killer cells, the generation of reactive oxygen species and nitric oxide from macrophages and the levels of Th1 regulatory cytokines remained unaltered. Interestingly, curcumin-injections enhanced the mitogen and antigen induced proliferation potential of T cells. We have also evaluated immunomodulatory effects of curcumin in ascites-bearing animals. This study strengthens our belief that curcumin is a safe and useful immunomodulator for the immune system.

STING activation enhances cetuximab-mediated NK cell activation and DC maturation and correlates with HPV+ status in head and neck cancer.

OBJECTIVES: The intracellular DNA sensor stimulator of interferon genes (STING) has recently been shown to play a vital role in anti-viral and anti-tumor immune responses stimulating cytokine production. While human papillomavirus (HPV) is a causative agent for a subset of head and neck squamous cell carcinoma (HNSCC) with unique etiology and clinical outcome, how the STING pathway is regulated in a virus-induced tumor microenvironment is not well understood. Since STING inactivation likely reflects immunoescape via innate immunity, we hypothesized that its restoration would improve efficacy of the immune modulatory monoclonal antibody (mAb), cetuximab. MATERIALS AND METHODS: We correlated STING protein expression with clinical parameters by immunohistochemistry (n = 106) and its mRNA expression from The Cancer Genome Atlas (TCGA) in HNSCC tissue specimens. STING protein expression was tested for association with cancer-specific survival (CSS). We further examined the impact of STING activation on cetuximab-mediated immunity using an in vitro NK:DC:tumor cells co-culture system. RESULTS: In this study, we found that expression of STING both at the protein and mRNA level was higher in HPV positive (HPV+) specimens but unrelated to TNM stage or cancer-specific survival. Our in vitro studies verified that STING activation enhanced cetuximab mediated NK cell activation and DC maturation. CONCLUSION: Our findings suggest a novel role of STING in HPV-related carcinogenesis, in which activation of the STING signaling pathway may facilitate anti-tumor response in HNSCC patients, particularly in combination with therapeutic monoclonal antibodies (mAbs) such as cetuximab, an epidermal growth factor receptor (EGFR) inhibitor.

Novel Effector Phenotype of Tim-3+ Regulatory T Cells Leads to Enhanced Suppressive Function in Head and Neck Cancer Patients.

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear.Experimental Design: CD4+CTLA-4+CD25high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T-cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs.Conclusions: Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Clin Cancer Res; 24(18); 4529-38. ©2018 AACR.

Phase Ib Study of Immune Biomarker Modulation with Neoadjuvant Cetuximab and TLR8 Stimulation in Head and Neck Cancer to Overcome Suppressive Myeloid Signals.

Purpose: The response rate of patients with head and neck squamous cell carcinoma (HNSCC) to cetuximab therapy is only 15% to 20%, despite frequent EGFR overexpression. Because immunosuppression is common in HNSCC, we hypothesized that adding a proinflammatory TLR8 agonist to cetuximab therapy might result in enhanced T-lymphocyte stimulation and anti-EGFR-specific priming.Experimental Design: Fourteen patients with previously untreated HNSCC were enrolled in this neoadjuvant trial and treated preoperatively with 3 to 4 weekly doses of motolimod (2.5 mg/m2) and cetuximab. Correlative tumor and peripheral blood specimens were obtained at baseline and at the time of surgical resection and analyzed for immune biomarker changes. Preclinical in vitro studies were also performed to assess the effect of cetuximab plus motolimod on myeloid cells.Results: TLR8 stimulation skewed monocytes toward an M1 phenotype and reversed myeloid-derived suppressor cell (MDSC) suppression of T-cell proliferation in vitro These data were validated in a prospective phase Ib neoadjuvant trial, in which fewer MDSC and increased M1 monocyte infiltration were found in tumor-infiltrating lymphocytes. Motolimod plus cetuximab also decreased induction of Treg and reduced markers of suppression, including CTLA-4, CD73, and membrane-bound TGFß. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhanced CD8+ T-cell infiltration into tumors. These T cells manifested increased T-cell receptor (TCR) clonality, upregulation of the costimulatory receptor CD27, and downregulation of inhibitory receptor TIGIT.Conclusions: Enhanced inflammatory stimulation in the tumor microenvironment using a TLR agonist overcomes suppressive myeloid and regulatory cells, enhancing the cellular antitumor immune response by therapeutic mAb in HNSCC. Clin Cancer Res; 24(1); 62-72. ©2017 AACR.