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Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103 + CD39 + tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103 + CD39 + CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103 + CD39 + CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103 + CD39 + CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies. Identifying and enumerating tumor-specific CD8 T cells are important for assessing cancer prognosis and therapy efficacy. Here the authors show that CD39 and CD103 mark a subset of tumor-infiltrating CD8 T cells that are tumor-reactive and exhibit characteristics of exhausted or tissue-resident memory T cells.

Blockade of NKG2A/HLA-E interaction is a promising strategy to unleash the anti-tumor response. Yet the role of NKG2A + CD8 T cells in the anti-tumor response and the regulation of NKG2A expression on human tumor-infiltrating T cells are still poorly understood. Here, by performing CITE-seq on T cells derived from head and neck squamous cell carcinoma and colorectal cancer, we show that NKG2A expression is induced on CD8 T cells differentiating into cytotoxic, CD39 + CD103 + double positive (DP) cells, a phenotype associated with tumor-reactive T cells. This developmental trajectory leads to TCR repertoire overlap between the NKG2A – and NKG2A + DP CD8 T cells, suggesting shared antigen specificities. Mechanistically, IL-12 is essential for the expression of NKG2A on CD8 T cells in a CD40/CD40L- dependent manner, in conjunction with TCR stimulation. Our study thus reveals that NKG2A is induced by IL-12 on human tumor-reactive CD8 T cells exposed to a TGF-β-rich environment, highlighting an underappreciated immuno-regulatory feedback loop dependent on IL-12 stimulation. Effective strategies to enhance T cell anti-tumor cytotoxicity are pivotal to improve treatment outcomes. By analyzing tumor samples from patients with head and neck or colon cancers, here the authors show that IL-12 can induce the expression of the inhibitory receptor NKG2A on tumor-reactive CD8 cytotoxic lymphocytes.

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.

Background Adoptive T cell therapy (ACT) has shown great promise in melanoma, with over 50 % response rate in patients where autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) can be cultured and expanded. A major limitation of ACT is the inability to generate or expand autologous tumor-reactive TIL in 25–45 % of patients tested. Methods that successfully identify tumors that are not suitable for TIL generation by standard methods would eliminate the costs of fruitless expansion and enable these patients to receive alternate therapy immediately. Methods Multispectral fluorescent immunohistochemistry with a panel including CD3, CD8, FoxP3, CD163, PD-L1 was used to analyze the tumor microenvironment in 17 patients with melanoma among our 36-patient cohort to predict successful TIL generation. Additionally, we compared tumor fragments and enzymatic digestion of tumor samples for efficiency in generating tumor-reactive TIL. Results Tumor-reactive TIL were generated from 21/36 (58 %) of melanomas and for 12/13 (92 %) tumors where both enzymatic and fragment methods were compared. TIL generation was successful in 10/13 enzymatic preparations and in 10/13 fragment cultures; combination of both methods resulted in successful generation of autologous tumor-reactive TIL in 12/13 patients. In 17 patients for whom tissue blocks were available, IHC analysis identified that while the presence of CD8 + T cells alone was insufficient to predict successful TIL generation, the CD8 + to FoxP3 + ratio was predictive with a positive-predictive value (PPV) of 91 % and negative-predictive value (NPV) of 86 %. Incorporation of CD163+ macrophage numbers and CD8:PD-L1 ratio did not improve the PPV. However, the NPV could be improved to 100 % by including the ratio of CD8 + :PD-L1 + expressing cells. Conclusion This is the first study to apply 7-color multispectral immunohistochemistry to analyze the immune environment of tumors from patients with melanoma. Assessment of the data using unsupervised hierarchical clustering identified tumors from which we were unable to generate TIL. If substantiated, this immune profile could be applied to select patients for TIL generation. Additionally, this biomarker profile may also indicate a pre-existing immune response, and serve as a predictive biomarker of patients who will respond to checkpoint blockade. We postulate that expanding the spectrum of inhibitory cells and molecules assessed using this technique could guide combination immunotherapy treatments and improve response rates.

The COVID-19 pandemic has created a high demand on personal protective equipment, including disposable N95 masks. Given the need for mask reuse, we tested the feasibility of vaporized hydrogen peroxide (VHP), ultraviolet light (UV), and ethanol decontamination strategies on N95 mask integrity and the ability to remove the infectious potential of SARS-CoV-2. Disposable N95 masks, including medical grade (1860, 1870+) and industrial grade (8511) masks, were treated by VHP, UV, and ethanol decontamination. Mask degradation was tested using a quantitative respirator fit testing. Pooled clinical samples of SARS-CoV-2 were applied to mask samples, treated, and then either sent immediately for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) or incubated with Vero E6 cells to assess for virucidal effect. Both ethanol and UV decontamination showed functional degradation to different degrees while VHP treatment showed no significant change after two treatments. We also report a single SARS-CoV-2 virucidal experiment using Vero E6 cell infection in which only ethanol treatment eliminated detectable SARS-CoV-2 RNA. We hope our data will guide further research for evidenced-based decisions for disposable N95 mask reuse and help protect caregivers from SARS-CoV-2 and other pathogens.

BackgroundBy integrating genomics, transcriptomics, proteomics, and metabolomics, researchers have gained unprecedented insights into the complex interplay between tumors and the immune system. This wealth of data has fueled the discovery of novel biomarkers for early cancer detection, prognostic stratification, and prediction of treatment response. Ultrahigh-plex multispectral immunohistochemistry enables the ability to analyze a large set of biomarkers simultaneously while maintaining the context of tissue architecture. By obtaining spatial information, researchers can understand the contact interactions and proximity between different cell types, leading to a deeper understanding of cellular dynamics and disease progression. This approach enables the identification of complex cellular patterns, such as immune cell infiltration, cell-cell interactions, or tumor heterogeneity. Ultimately, these findings may contribute to the development of more effective therapies and diagnostics for cancer patients.MethodsWe performed whole-slide spatial phenotyping with an ultrahigh-plex PhenoCycler-Fusion panel on FFPE human head and neck tumor biopsies (pre-treatment and post-treatment) from a patient receiving immunotherapy. Enumeration of cell phenotypes and spatial location analysis was performed using QuPath,1 R, and proprietary software.ResultsWithin the invasive margin of the tumor (+/- 20mm of the tumor edge) we measured baseline densities of CD8+ T cells (264 cells/mm2) and CD4+ T cells (458 cells/mm2) at the pretreatment timepoint, with the densities substantially increased following treatment (1042 cells/mm2 CD8; 652 cells/mm2 CD4). The CD8+ T cells present within the invasive margin also displayed functionality as evidenced by their expression of GranzymeB (109 vs 397 cells/mm2, pre vs post). In addition, differential enrichment was observed within the invasive margin compared to the entire biopsy (41% vs 58%, pre vs post). In conjunction tumor cells at the post-treatment timepoint showed higher expression of the pro-apoptotic BAX protein (34% vs 58%, pre vs post) with reduced expression of the anti-apoptotic Bcl-2 protein (32% vs 12%, pre vs post).ConclusionsData from this ultrahigh-plex multispectral panel has provided a number of potential biomarkers for investigation in patients with head and neck cancer receiving immunotherapy. These findings inform development of smaller, more efficient multispectral panels to be designed and run on larger cohorts of patients. Additionally, examining cell populations residing within specific spatial tumor microenvironments (ie., invasive margin) uncovers additional metrics that should be examined in association with a patient’s response to treatment.AcknowledgementsSupport: Providence Medical Foundation, The Chiles Foundation, The Harder Family, Nancy Lematta, Robert W. Franz and Elsie Franz Finley, The Murdoch Memorial TrustReferenceBankhead P, et al. QuPath: Open source software for digital pathology image analysis. Scientific Reports 2017.Ethics ApprovalPSJH IRB# 06–108

BackgroundShort-lived proteins (SLiPs), defective ribosomal products (DRiPs), and Cancer’s dark matter/non-canonical peptides (NCP) are unstable and rapidly degraded, loaded onto MHC and represent a large proportion of the epitopes presented by cancer cells. Recent data suggests that NCP may play a role in molecular control of some malignant processes, further strengthening the rationale for characterizing their presence and developing pharmacologic and immunologic approaches to impact their effect.1 Immunologic approaches seem particularly promising as NCP, that are not expressed in the thymus, represent potential shared alternative cancer neoantigens.2 Our group developed a vaccine strategy that concentrates SLiPs, DRiPs, and NCP in dendritic cell-targeted microvesicles.3 Preclinical combination immunotherapy studies documented efficacy in difficult to treat animal models4 5 and an off-the-shelf human vaccine was developed and has entered clinical trials. The current studies were undertaken to characterize the breadth of peptides presented by head and neck squamous cell cancer (HNSCC) and non-small cell lung cancer (NSCLC), as well as the proteins contained in the DPV-001 vaccine.MethodsCancer cells were lysed and recovered lysates were treated with detergent in the presence of protease inhibitor. HLA peptides were collected from HLA complexes purified by anti-HLA-I antibody (w6/32). Recovered HLA peptides were analyzed by the microLC-QTOF MS (LCMS-9030. Shimadzu Corporation). The two components of DPV-001, UbiLT3 and UbiLT6, were assessed via deep proteomic profiling on an Orbitrap mass spectrometer (ORBITRAP FUSION LUMOS with FAIMS-Pro interface (Thermofisher Scientific)). Spectra from whole proteome tryptic digests were acquired in data-dependent acquisition (DDA) mode, and Peptide-Prism was used to identify canonical and noncanonical peptides/NCPs.ResultsThe approaches outlined above provide evidence of canonical and NCP, derived from noncoding or out-of-frame translations, presented by HLA of HNSCC cell lines and contained within DPV-001. These peptides provide a starting point for immunological studies.ConclusionsThese results support our efforts to identify shared canonical and NCP that are targets of therapeutic immune responses. The discovery of NCP derived from supposed non-coding regions that are not expressed in the thymus, represent a novel class of potentially shared alternative cancer neoantigens. Their association with malignant processes may provide them properties similar to that of driver mutations, and increase their relevance as cancer vaccine targets.AcknowledgementsSupport: Shimadzu Corporation, Providence Medical Foundation, The Harder Family, Nancy Lematta, The Chiles Foundation, Robert W. Franz, and Elsie Franz FinleyReferencesFox BA, Urba WJ, Jensen SM, Page DB, Curti BD, Sanborn RE, Leidner RS. Cancer’s Dark Matter: Lighting the Abyss Unveils Universe of New Therapies. Clinical Cancer Research 2023; 29:2173–2175. 10.1158/1078–0432.CCR-23–0422.Cieri N, Wu CJ. Splice it up: Atypical transcripts to boost leukemia immunotherapy. Immunity 2021; 54:608–610. 10.1016/j.immuni.2021.03.016.Page DB, Hulett TW, Hilton TL, Hu H-M, Urba WJ, Fox BA. Glimpse into the future: harnessing autophagy to promote anti-tumor immunity with the DRibbles vaccine. Journal for ImmunoTherapy of Cancer 2016; 4:10.1186/s40425–016-0130–4.Yu G, Li Y, Cui Z, Morris NP, Weinberg AD, Fox BA, Urba WJ, Wang L, Hu H-M. Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression. Scientific Reports 2016; 6:10.1038/srep37558.Patel JM, Cui Z, Wen Z-F, Dinh CT, Hu H-M. Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism. J Immunother Cancer 2019; 7:311. 10.1186/s40425–019-0786–7.Ethics ApprovalPSJH IRB# 06–108

BackgroundOur group developed a strategy for generating an ‘off-the-shelf’ multivalent proteasome-blocked autophagosome vaccine that contains proteins for many genes commonly overexpressed in adenocarcinoma and squamous cell cancers. This strategy exploits in vitro manipulation of the antigen presentation pathway to concentrate the dominant epitopes presented by MHC, including short-lived proteins (SLiPs), defective ribosomal products (DRiPs), and Dark Matter, the short-lived non-canonical peptides that are not expressed in the thymus and represent potential shared alternative cancer neoantigens.1 In preclinical models this vaccine strategy provides significant protection as a single agent,2 and significantly increased therapeutic efficacy when combined with anti-GITR and anti-PD-1.3 Based on these studies we hypothesize that addition of anti-GITR to the human vaccine, DPV-001, and anti-PD-1, will augment expansion and limit contraction of the anti-cancer immune response. A phase I clinical trial was initiated, and preliminary immunological monitoring data will be presented.MethodsPatients received DPV-001, with sequenced checkpoint inhibition (aPD-1 mAb; retifanlimab), with or without aGITR agonist mAb (INCAGN1876), in recurrent or metastatic HNSCC (NCT04470024). Tumor biopsies were taken pre-treatment, week 2 and 8. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Tumor biopsies and blood were assayed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluorescence (mIF) was performed on biopsies.ResultsIn the first 4 patients evaluated, tumor-infiltrating T cells at week 8 increased from pre-treatment levels by an average of 4.3 fold (range 2.9 – 6.7, p=0.032). The density of CD39/CD103 double positive cells, previously shown to identify tumor-reactive T cells,4 also increased in all week 8 biopsies (mean 14.7 fold, range 5–40). All patients showed increased numbers of cells expressing IFN-γ and GZMB and increased numbers of T cells expressing LAG3+ in week 8 biopsies. Preliminary TCR evaluation of the tumor identified proliferation of clones previously undetected in PBL, including αβ T cells, iNKT and MAIT cells. Expansion of clones that predated treatment was also identified.ConclusionsAn increase in intra-tumoral T cells expressing activation and effector molecules is encouraging and studies are underway to expand the number of patients analyzed. Increased expression of LAG3 by T cells that infiltrate and have expanded in the tumor, provide a rationale for including anti-LAG-3 in this treatment strategy. Future plans include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001 and whether antibody responses identify targets of cellular immunity.AcknowledgementsSupport: Incyte, UbiVac, Providence Medical Foundation, The Chiles Foundation, The Harder Family, Nancy Lematta, Robert W. Franz and Elsie Franz Finley, The Murdoch Memorial TrustTrial RegistrationNCT04470024ReferencesFox BA, Urba WJ, Jensen SM, Page DB, Curti BD, Sanborn RE, Leidner RS. Cancer’s Dark Matter: Lighting the Abyss Unveils Universe of New Therapies. Clinical Cancer Research 2023; 29:2173–2175. 10.1158/1078–0432.CCR-23–0422.Twitty CG, Jensen SM, Hu H-M, Fox BA. Tumor-Derived Autophagosome Vaccine: Induction of Cross-Protective Immune Responses against Short-lived Proteins through a p62-Dependent Mechanism. Clinical Cancer Research 2011; 17:6467–6481. 10.1158/1078–0432.CCR-11–0812.Patel JM, Cui Z, Wen Z-F, Dinh CT, Hu H-M. Peritumoral administration of DRibbles-pulsed antigen-presenting cells enhances the antitumor efficacy of anti-GITR and anti-PD-1 antibodies via an antigen presenting independent mechanism. J Immunother Cancer 2019; 7:311. 10.1186/s40425–019-0786–7.Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018; 9:2724. 10.1038/s41467–018-05072–0.Ethics ApprovalPSJH IRB# 2020000480

BackgroundTumor Infiltrating Lymphocyte (TIL) therapy has proven effective for patients with stage IV melanoma, however there are critical issues that can limit the efficacy of standard TIL therapy across a wide range of different malignancies. We and others have shown that some tumor types contain a low percentage of tumor-specific T cells. We hypothesize that most of the patients that do not respond to TIL therapy are likely receiving a low percentage of tumor-reactive T cells and therefore a high percentage of non-therapeutic bystander TIL. We have developed a streamlined method that expands a highly enriched fraction of tumor-reactive T cells contained within the CD39+CD103+CD8+ TIL in greater than 90% of patient samples from a wide variety of malignancies (melanoma, colon cancer, head and neck cancer, etc.). This TIL product displays a broad repertoire of tumor-specific TCRs. The expanded CD39/CD103 TIL can kill autologous tumors in vitro, but the possibility remains that they could revert to a suppressed or exhausted state when they reach the tumor microenvironment upon transfer back into patients. To mitigate the suppressive effects of the tumor microenvironment we have evaluated Phio Pharmaceutical’s self-delivering RNAi INTASYL(TM) platform to silence PD-1 in the expanded TIL product.MethodsThe TIL product was treated during the rapid expansion phase of the protocol with either nontargeting control compounds or PD-1 targeting INTASYL™ compounds. PD-1 protein levels and TIL functionality were assessed via flow cytometry and cytokine bead array.ResultsSilencing of PD-1 expression in the expanded TIL product was obtained by adding the self-delivering RNAi compounds to the cell culture media, without needing transfection media, delivery formulations or electroporation. The RNAi-treated TIL product showed increased IFN-?? TNF-α and Granzyme B expression.ConclusionsThese data highlight a promising combination to improve the activity of tumor-reactive TIL in future human clinical trials.

BackgroundOutcomes for recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are dismal and responses to anti-PD-1 appear best in tumors with PD-1+ T cells in proximity to PD-L1+ cells, arguing that improved outcome is associated with a pre-existing anti-cancer immune response. Based on this, we hypothesize that vaccines which prime and/or expand T cells to a spectrum of antigens overexpressed by HNSCC combined with T cell agonists, like anti-GITR, that provide costimulatory signals will improve the anti-PD-1 response rates. We have developed a cancer vaccine, DPV-001, that contains more than 300 proteins for genes overexpressed by HNSCC, encapsulated in a CLEC9A-targeted microvesicle and containing TLR/NOD agonists and DAMPs. Recently, we reported that combining anti-GITR + vaccine + anti-PD-1 augmented therapeutic efficacy in a preclinical model and now plan a phase 1b trial of this combination in patients with advanced HNSCC.MethodsSera from patients receiving DPV-001 as adjuvant therapy for definitively treated NSCLC, were analyzed for IgG responses to human proteins by MAP bead arrays and results compared to TCGA gene expression data sets for HNSCC. HNSCC cell lines were evaluated by RNASeq and peptides were eluted from HLA, analyzed by mass spectroscopy and correlated against MAP bead arrays and TCGA data sets. Tumor-reactive T cells from a vaccinated patient were enriched and expanded, and used in cytokine release assay (CRA) against autologous NSCLC and partially HLA matched allogeneic HNSCC cell lines.ResultsPatients receiving DPV-001 (N=13) made 147 IgG responses to at least 70 proteins for genes overexpressed by HNSCC. Preliminary evaluation of the HNSCC peptidome against the results of MAP bead array identify antigens that are target of a humoral immune response. Additionally, tumor-reactive T cells from DPV-001 vaccinated patient recognize two partially HLA-matched HNSCC targets, but not a mis-matched target.ConclusionsRecent observations from our lab and others have correlated IgG Ab responses with T cell responses to epitopes of the same protein. Based on the data summarized above, we hypothesize that we have induced T cell responses against a broad spectrum of shared cancer antigens that are common among adenocarcinomas and squamous cell cancers. Our planned clinical trial will vaccinate and boost the induced responses by costimulation with anti-GITR and then sequence in delayed anti-PD-1 to relieve checkpoint inhibition. MAP bead arrays and the peptidome library generated above will be used to assess anti-cancer B and T cell responses.Trial RegistrationNCT04470024Ethics ApprovalThe original clinical trial was approved by the Providence Portland Medical Center IRB, approval # 13-046. The proposed clinical trial has not yet been reviewed by the IRB.