Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.

Recent advances in multiplex immunohistochemistry techniques allow for quantitative, spatial identification of multiple immune parameters for enhanced diagnostic and prognostic insight. However, applying such techniques to murine fixed tissues, particularly sensitive epitopes, such as CD4, CD8α, and CD19, has been difficult. We compared different fixation protocols and Ag-retrieval techniques and validated the use of multiplex immunohistochemistry for detection of CD3(+)CD4(+) and CD3(+)CD8(+) T cell subsets in murine spleen and tumor. This allows for enumeration of these T cell subsets within immune environments, as well as the study of their spatial distribution.

Tumor-specific CD4+ T cells maintain effector and memory tumor-specific CD8+ T cells.

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor-specific CD4(+) T cells enhance CD8(+) T-cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase-related protein 1-specific CD4(+) transgenic T cells-CD4(+) T cells and pmel-CD8(+) T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8(+) T cells with tumor-specific cytokine expression. When combined with CD4(+) T cells, transfer of total (naïve and effector) or effector CD8(+) T cells were highly effective, suggesting CD4(+) T cells can help mediate therapeutic effects by maintaining function of activated CD8(+) T cells. In addition, CD4(+) T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8(+) T cells recovered from mice treated with both CD8(+) and CD4(+) T cells had decreased expression of PD-1 and PD-1-blockade enhanced the therapeutic efficacy of pmel-CD8 alone, suggesting that CD4(+) T cells help reduce CD8(+) T-cell exhaustion. These data support combining immunotherapies that elicit both tumor-specific CD4(+) and CD8(+) T cells for treatment of patients with cancer.

Increased frequency of suppressive regulatory T cells and T cell-mediated antigen loss results in murine melanoma recurrence.

Therapeutic treatment of large established tumors using immunotherapy has yielded few promising results. We investigated whether adoptive transfer of tumor-specific CD8(+) T cells, together with tumor-specific CD4(+) T cells, would mediate regression of large established B16BL6-D5 melanomas in lymphopenic Rag1(-/-) recipients devoid of regulatory T cells. The combined adoptive transfer of subtherapeutic doses of both TRP1-specific TCR transgenic Rag1(-/-) CD4(+) T cells and gp100-specific TCR transgenic Rag1(-/-) CD8(+) T cells into lymphopenic recipients, who received vaccination, led to regression of large (100-400 mm(2)) melanomas. The same treatment strategy was ineffective in lymphoreplete wild-type mice. Twenty-five percent of mice (15/59) had tumors recur (15-180 d postregression). Recurrent tumors were depigmented and had decreased expression of gp100, the epitope targeted by the CD8(+) T cells. Mice with recurrent melanoma had increased CD4(+)Foxp3(+) TRP1-specific T cells compared with mice that did not show evidence of disease. Importantly, splenocytes from mice with recurrent tumor were able to suppress the in vivo therapeutic efficacy of splenocytes from tumor-free mice. These data demonstrate that large established tumors can be treated by a combination of tumor-specific CD8(+) and CD4(+) T cells. Additionally, recurrent tumors exhibited decreased Ag expression, which was accompanied by conversion of the therapeutic tumor-specific CD4(+) T cell population to a Foxp3(+)CD4(+) regulatory T cell population.

FcγRIIB Is an Immune Checkpoint Limiting the Activity of Treg-Targeting Antibodies in the Tumor Microenvironment.

Preclinical murine data indicate that fragment crystallizable (Fc)-dependent depletion of intratumoral regulatory T cells (Treg) is a major mechanism of action of anti-CTLA-4. However, the two main antibodies administered to patients (ipilimumab and tremelimumab) do not recapitulate these effects. Here, we investigate the underlying mechanisms responsible for the limited Treg depletion observed with these therapies. Using an immunocompetent murine model humanized for CTLA-4 and Fcγ receptors (FcγR), we show that ipilimumab and tremelimumab exhibit limited Treg depletion in tumors. Immune profiling of the tumor microenvironment (TME) in both humanized mice and humans revealed high expression of the inhibitory Fc receptor, FcγRIIB, which limits antibody-dependent cellular cytotoxicity/phagocytosis. Blocking FcγRIIB in humanized mice rescued the Treg-depleting capacity and antitumor activity of ipilimumab. Furthermore, Fc engineering of antibodies targeting Treg-associated targets (CTLA-4 or CCR8) to minimize FcγRIIB binding significantly enhanced Treg depletion, resulting in increased antitumor activity across various tumor models. Our results define the inhibitory FcγRIIB as an immune checkpoint limiting antibody-mediated Treg depletion in the TME, and demonstrate Fc engineering as an effective strategy to overcome this limitation and improve the efficacy of Treg-targeting antibodies.

Treatment of malignant pleural mesothelioma by fibroblast activation protein-specific re-directed T cells.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an incurable malignant disease, which results from chronic exposition to asbestos in at least 70% of the cases. Fibroblast activation protein (FAP) is predominantly expressed on the surface of reactive tumor-associated fibroblasts as well as on particular cancer types. Because of its expression on the cell surface, FAP is an attractive target for adoptive T cell therapy. T cells can be re-directed by retroviral transfer of chimeric antigen receptors (CAR) against tumor-associated antigens (TAA) and therefore represent a therapeutic strategy of adoptive immunotherapy. METHODS: To evaluate FAP expression immunohistochemistry was performed in tumor tissue from MPM patients. CD8+ human T cells were retrovirally transduced with an anti-FAP-F19-∆CD28/CD3ζ-CAR. T cell function was evaluated in vitro by cytokine release and cytotoxicity assays. In vivo function was tested with an intraperitoneal xenograft tumor model in immunodeficient mice. RESULTS: FAP was found to be expressed in all subtypes of MPM. Additionally, FAP expression was evaluated in healthy adult tissue samples and was only detected in specific areas in the pancreas, the placenta and very weakly for cervix and uterus. Expression of the anti-FAP-F19-∆CD28/CD3ζ-CAR in CD8+ T cells resulted in antigen-specific IFNγ release. Additionally, FAP-specific re-directed T cells lysed FAP positive mesothelioma cells and inflammatory fibroblasts in an antigen-specific manner in vitro. Furthermore, FAP-specific re-directed T cells inhibited the growth of FAP positive human tumor cells in the peritoneal cavity of mice and significantly prolonged survival of mice. CONCLUSION: FAP re-directed CD8+ T cells showed antigen-specific functionality in vitro and in vivo. Furthermore, FAP expression was verified in all MPM histotypes. Therefore, our data support performing a phase I clinical trial in which MPM patients are treated with adoptively transferred FAP-specific re-directed T cells.

Cancer's Dark Matter: Lighting the Abyss Unveils Universe of New Therapies.

The authors of a recent study identified noncanonical peptides (NCP) presented by cancer cells' HLA and observed lack of reactivity to these antigens by endogenous tumor-reactive T cells. In vitro sensitization generated NCP-reactive T cells that recognized epitopes shared by a majority of cancers tested, providing opportunities for novel therapies to shared antigens. See related article by Lozano-Rabella et al., p. 2250.

FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies.

Despite pre-clinical murine data supporting T regulatory (Treg) cell depletion as a major mechanism by which anti-CTLA-4 antibodies function in vivo, the two main antibodies tested in patients (ipilimumab and tremelimumab) have failed to demonstrate similar effects. We report analogous findings in an immunocompetent murine model humanized for CTLA-4 and Fcy receptors (hCTLA-4/hFcyR mice), where both ipilimumab and tremelimumab fail to show appreciable Treg depletion. Immune profiling of the tumor microenvironment (TME) in both mice and human samples revealed upregulation of the inhibitory Fcy receptor, FcyRIIB, which limits the ability of the antibody Fc fragment of human anti-CTLA-4 antibodies to induce effective antibody dependent cellular cytotoxicty/phagocytosis (ADCC/ADCP). Blocking FcyRIIB in humanized mice rescues Treg depleting capacity and anti-tumor activity of ipilimumab. For another target, CC motif chemokine receptor 8 (CCR8), which is selectively expressed on tumor infiltrating Tregs, we show that Fc engineering to enhance binding to activating Fc receptors, while limiting binding to the inhibitory Fc receptor, leads to consistent Treg depletion and single-agent activity across multiple tumor models, including B16, MC38 and MB49. These data reveal the importance of reducing engagement to the inhibitory Fc receptor to optimize Treg depletion by TME targeting antibodies. Our results define the inhibitory FcyRIIB receptor as a novel immune checkpoint limiting antibody-mediated Treg depletion in tumors, and demonstrate Fc variant engineering as a means to overcome this limitation and augment efficacy for a repertoire of antibodies currently in use or under clinical evaluation in oncology.

OX40 agonist stimulation increases and sustains humoral and cell-mediated responses to SARS-CoV-2 protein and saRNA vaccines.

To prevent SARS-CoV-2 infections and generate long-lasting immunity, vaccines need to generate strong viral-specific B and T cell responses. Previous results from our lab and others have shown that immunizations in the presence of an OX40 agonist antibody lead to higher antibody titers and increased numbers of long-lived antigen-specific CD4 and CD8 T cells. Using a similar strategy, we explored the effect of OX40 co-stimulation in a prime and boost vaccination scheme using an adjuvanted SARS-CoV-2 spike protein vaccine in C57BL/6 mice. Our results show that OX40 engagement during vaccination significantly increases long-lived antibody responses to the spike protein. In addition, after immunization spike protein-specific proliferation was greatly increased for both CD4 and CD8 T cells, with enhanced, spike-specific secretion of IFN-γ and IL-2. Booster (3rd injection) immunizations combined with an OX40 agonist (7 months post-prime) further increased vaccine-specific antibody and T cell responses. Initial experiments assessing a self-amplifying mRNA (saRNA) vaccine encoding the spike protein antigen show a robust antigen-specific CD8 T cell response. The saRNA spike-specific CD8 T cells express high levels of GrzmB, IFN-γ and TNF-α which was not observed with protein immunization and this response was further increased by the OX40 agonist. Similar to protein immunizations the OX40 agonist also increased vaccine-specific CD4 T cell responses. In summary, this study compares and contrasts the effects and benefits of both protein and saRNA vaccination and the extent to which an OX40 agonist enhances and sustains the immune response against the SARS-CoV-2 spike protein.

Lymphatic-preserving treatment sequencing with immune checkpoint inhibition unleashes cDC1-dependent antitumor immunity in HNSCC.

Despite the promise of immune checkpoint inhibition (ICI), therapeutic responses remain limited. This raises the possibility that standard of care treatments delivered in concert may compromise the tumor response. To address this, we employ tobacco-signature head and neck squamous cell carcinoma murine models in which we map tumor-draining lymphatics and develop models for regional lymphablation with surgery or radiation. We find that lymphablation eliminates the tumor ICI response, worsening overall survival and repolarizing the tumor- and peripheral-immune compartments. Mechanistically, within tumor-draining lymphatics, we observe an upregulation of conventional type I dendritic cells and type I interferon signaling and show that both are necessary for the ICI response and lost with lymphablation. Ultimately, we provide a mechanistic understanding of how standard oncologic therapies targeting regional lymphatics impact the tumor response to immune-oncology therapy in order to define rational, lymphatic-preserving treatment sequences that mobilize systemic antitumor immunity, achieve optimal tumor responses, control regional metastatic disease, and confer durable antitumor immunity.

Disruption of TGF-beta signaling prevents the generation of tumor-sensitized regulatory T cells and facilitates therapeutic antitumor immunity.

Regulatory T (Treg) cells represent a major roadblock to the induction of antitumor immunity through vaccine approaches. TGF-beta is a cytokine implicated in the generation and maintenance of Treg cells, as well as in their suppressive function. These experiments examined whether the generation of tumor-sensitized Treg cells was TGF-beta dependent and evaluated whether TGF-beta produced by Treg cells blocked the priming of tumor-specific T cells in vaccinated reconstituted lymphopenic mice. We show that tumor-sensitized Treg cells (CD25(+)/FoxP3(+)) obtained from tumor-bearing mice block the generation of tumor-specific T cells in reconstituted lymphopenic mice. Strikingly, this suppression is absent if tumor-sensitized Treg cells are acquired from tumor-bearing mice expressing the dominant-negative TGFbetaRII in T cells. This loss of suppression was a result of the crucial role of TGF-beta in generating tumor-sensitized Treg cells, and not due to the insensitivity of naive or tumor-primed effector T cells to the direct suppressive influence of TGF-beta. We conclude that blocking TGF-beta in a tumor-bearing host can inhibit the induction of highly suppressive tumor-sensitized Treg cells. These data suggest that an integrative strategy combining "up-front" Treg cell ablation followed by vaccination and TGF-beta blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.

SARS-CoV-2 Antibodies Detected in Mother's Milk Post-Vaccination.

BACKGROUND: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has infected over 127 million people worldwide, with almost 2.8 million deaths at the time of writing. Since no lactating individuals were included in initial trials of vaccine safety and efficacy, research on SARS-CoV-2 vaccination in lactating women and the potential transmission of passive immunity to the infant through mother's milk is needed to guide patients, clinicians, and policy makers on whether to recommend immunization during the worldwide effort to curb the spread of this virus. RESEARCH AIMS: (1) To determine whether SARS-CoV-2 specific immunoglobins are found in human milk after vaccination, and (2) to characterize the time course and types of immunoglobulins present. METHODS: A longitudinal cohort study of lactating women (N = 7) who planned to receive both doses of the Pfizer-BioNTech or Moderna SARS-CoV-2 vaccine between December 2020 and January 2021 provided milk samples. These were collected pre-vaccination and at 11 additional timepoints, with the last sample at 14 days after the second dose of vaccine. Samples were analyzed for levels of SARS-CoV-2 specific immunoglobulins A and G (IgA and IgG). RESULTS: We observed significantly elevated levels of SARS-CoV-2 specific IgG and IgA antibodies in human milk beginning approximately 7 days after the initial vaccine dose, with an IgG-dominant response. CONCLUSIONS: Maternal vaccination results in SARS-CoV-2 specific immunoglobulins in human milk that may be protective for infants.

Phosphorylation of SMAD3 in immune cells predicts survival of patients with early stage non-small cell lung cancer.

BACKGROUND: The interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment. METHODS: Human non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data. RESULTS: TGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer. CONCLUSIONS: TGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response.

A platform for locoregional T-cell immunotherapy to control HNSCC recurrence following tumor resection.

Surgical resection of head and neck squamous-cell carcinoma (HNSCC) is associated with high rates of local and distant recurrence, partially mitigated by adjuvant therapy. A pre-existing immune response in the patient's tumor is associated with better outcomes following treatment with conventional therapies, but improved options are needed for patients with poor anti-tumor immunity. We hypothesized that local delivery of tumor antigen-specific T-cells into the resection cavity following surgery would direct T-cells to residual antigens in the margins and draining lymphatics and present a platform for T-cell-targeted immunotherapy. We loaded T-cells into a biomaterial that conformed to the resection cavity and demonstrated that it could release T-cells that retained their functional activity in-vitro, and in a HNSCC model in-vivo. Locally delivered T-cells loaded in a biomaterial were equivalent in control of established tumors to intravenous adoptive T-cell transfer, and resulted in the systemic circulation of tumor antigen-specific T-cells as well as local accumulation in the tumor. We demonstrate that adjuvant therapy with anti-PD1 following surgical resection was ineffective unless combined with local delivery of T-cells. These data demonstrate that local delivery of tumor-specific T-cells is an efficient option to convert tumors that are unresponsive to checkpoint inhibitors to permit tumor cures.

Multi-institutional TSA-amplified Multiplexed Immunofluorescence Reproducibility Evaluation (MITRE) Study.

BACKGROUND: Emerging data suggest predictive biomarkers based on the spatial arrangement of cells or coexpression patterns in tissue sections will play an important role in precision immuno-oncology. Multiplexed immunofluorescence (mIF) is ideally suited to such assessments. Standardization and validation of an end-to-end workflow that supports multisite trials and clinical laboratory processes are vital. Six institutions collaborated to: (1) optimize an automated six-plex assay focused on the PD-1/PD-L1 axis, (2) assess intersite and intrasite reproducibility of staining using a locked down image analysis algorithm to measure tumor cell and immune cell (IC) subset densities, %PD-L1 expression on tumor cells (TCs) and ICs, and PD-1/PD-L1 proximity assessments. METHODS: A six-plex mIF panel (PD-L1, PD-1, CD8, CD68, FOXP3, and CK) was rigorously optimized as determined by quantitative equivalence to immunohistochemistry (IHC) chromogenic assays. Serial sections from tonsil and breast carcinoma and non-small cell lung cancer (NSCLC) tissue microarrays (TMAs), TSA-Opal fluorescent detection reagents, and antibodies were distributed to the six sites equipped with a Leica Bond Rx autostainer and a Vectra Polaris multispectral imaging platform. Tissue sections were stained and imaged at each site and delivered to a single site for analysis. Intersite and intrasite reproducibility were assessed by linear fits to plots of cell densities, including %PDL1 expression by TCs and ICs in the breast and NSCLC TMAs. RESULTS: Comparison of the percent positive cells for each marker between mIF and IHC revealed that enhanced amplification in the mIF assay was required to detect low-level expression of PD-1, PD-L1, FoxP3 and CD68. Following optimization, an average equivalence of 90% was achieved between mIF and IHC across all six assay markers. Intersite and intrasite cell density assessments showed an average concordance of R2=0.75 (slope=0.92) and R2=0.88 (slope=0.93) for breast carcinoma, respectively, and an average concordance of R2=0.72 (slope=0.86) and R2=0.81 (slope=0.68) for NSCLC. Intersite concordance for %PD-L1+ICs had an average R2 value of 0.88 and slope of 0.92. Assessments of PD-1/PD-L1 proximity also showed strong concordance (R2=0.82; slope=0.75). CONCLUSIONS: Assay optimization yielded highly sensitive, reproducible mIF characterization of the PD-1/PD-L1 axis across multiple sites. High concordance was observed across sites for measures of density of specific IC subsets, measures of coexpression and proximity with single-cell resolution.

Intratumoral Plasmid IL12 Expands CD8+ T Cells and Induces a CXCR3 Gene Signature in Triple-negative Breast Tumors that Sensitizes Patients to Anti-PD-1 Therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed. PATIENTS AND METHODS: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets. RESULTS: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8+ T-cell infiltration following treatment. One patient, previously unresponsive to anti-PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti-PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response. CONCLUSIONS: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC.

Heterodimeric IL-15 delays tumor growth and promotes intratumoral CTL and dendritic cell accumulation by a cytokine network involving XCL1, IFN-γ, CXCL9 and CXCL10.

BACKGROUND: Interleukin-15 (IL-15) promotes growth and activation of cytotoxic CD8+ T and natural killer (NK) cells. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and IL-15 receptor alpha chains (hetIL-15). Several preclinical models support the antitumor activity of hetIL-15 promoting its application in clinical trials. METHODS: The antitumor activity of hetIL-15 produced from mammalian cells was tested in mouse tumor models (MC38 colon carcinoma and TC-1 epithelial carcinoma). The functional diversity of the immune infiltrate and the cytokine/chemokine network within the tumor was evaluated by flow cytometry, multicolor immunohistochemistry (IHC), gene expression profiling by Nanostring Technologies, and protein analysis by electrochemiluminescence and ELISA assays. RESULTS: hetIL-15 treatment resulted in delayed primary tumor growth. Increased NK and CD8+ T cell tumoral infiltration with an increased CD8+/Treg ratio were found by flow cytometry and IHC in hetIL-15 treated animals. Intratumoral NK and CD8+ T cells showed activation features with enhanced interferon-γ (IFN-γ) production, proliferation (Ki67+), cytotoxic potential (Granzyme B+) and expression of the survival factor Bcl-2. Transcriptomics and proteomics analyses revealed complex effects on the tumor microenvironment triggered by hetIL-15 therapy, including increased levels of IFN-γ and XCL1 with intratumoral accumulation of XCR1+IRF8+CD103+ conventional type 1 dendritic cells (cDC1). Concomitantly, the production of the chemokines CXCL9 and CXCL10 by tumor-localized myeloid cells, including cDC1, was boosted by hetIL-15 in an IFN-γ-dependent manner. An increased frequency of circulating CXCR3+ NK and CD8+ T cells was found, suggesting their ability to migrate toward the tumors following the CXCL9 and CXCL10 chemokine gradient. CONCLUSIONS: Our results show that hetIL-15 administration enhances T cell entry into tumors, increasing the success rate of immunotherapy interventions. Our study further supports the incorporation of hetIL-15 in tumor immunotherapy approaches to promote the development of antitumor responses by favoring effector over regulatory cells and by promoting lymphocyte and DC localization into tumors through the modification of the tumor chemokine and cytokine milieu.

Effect of various decontamination procedures on disposable N95 mask integrity and SARS-CoV-2 infectivity.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Future Research Goals in Immunotherapy.

In our opinion the most urgent needs to improve patient outcomes are: 1) a deeper ability to measure cancer immunobiology, and 2) increased availability of agents that, coupled with predictive biomarkers, will be used to tailor anti-cancer immunity. Tailoring effective immunotherapy will entail combinations of immunotherapeutics that augment priming of anti-cancer immunity, boost expansion of effector and memory cells of the T, B and NK lineage, amplify innate immunity and relieve checkpoint inhibition. Alternatives to inducing adaptive immunity to cancer include synthetic immunology that incorporate bi-specifics that target T cells to cancer or adoptive immunotherapy with gene-modified immune cells.

Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells.

Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis.This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.