Cytotoxic T Lymphocyte Activity Research Articles

Histone deacetylase inhibition can revert induced resistance to immune checkpoint blockade in a mouse model of prostate cancer.

Abstract Castration resistant prostate cancer (CRPC) is one of the leading causes of death in men in the United States, with a 5-year survival rate of 28%. Immune checkpoint blockade (PD-1 & CTLA4 inhibition, ICB) is a novel therapeutic strategy based on restoring the tumoricidal activity of cytotoxic T-lymphocytes. ICB therapy has produced spectacular results in the treatment of melanoma but shows almost no effect in CRPC. We have developed a model system of advanced prostate cancer (Pten−/−/Tp53−/−/Smad4−/−) consisting of a parental (ICB sensitive) cell line and an ICB resistant subline (PPS-ICBR). We analyzed the transcriptome and found significant changes associated with ICB resistance. Subsequently we reversed some of those changes with Vorinostat, a histone deacetylase inhibitor. Mice injected subcutaneously with the PPS-ICBR cell line showed no response to Vorinostat or ICB therapy, but when combined 40% of the mice were completely cured, and another 40% showed significant reductions in tumor growth. We dissociated the tumors and used mass cytometry to determine the changes in the tumor immune microenvironment. ICB + Vorinostat therapy causes a significant increase in CD4+ & CD8+ T-cells, and a significant decrease in M2 macrophages. Concurrent with this decrease in M2 macrophages we observed a significant increase of TNFα+/iNOS+ monocyte derived dendritic cells. Our goal is to study the underlying mechanisms that can transdifferentiate an immuno-suppressive tumor immune microenvironment towards a tumor-suppressive immune microenvironment and improve clinical outcomes.

Durvalumab Combined with Immunomodulatory Drugs (IMiD) Overcomes Suppression of Antitumor Responses due to IMiD-induced PD-L1 Upregulation on Myeloma Cells.

We previously showed that the interaction of programmed death-ligand 1 (PD-L1) on multiple myeloma (MM) cells with PD-1 not only inhibits tumor-specific cytotoxic T-lymphocyte activity via the PD-1 signaling pathway but also induces drug resistance via PD-L1-mediated reverse signals. We here examined the regulation of PD-L1 expression by immunomodulatory drugs (IMiDs) and antimyeloma effects of the anti-PD-L1 antibody durvalumab in combination with IMiDs. IMiDs induced PD-L1 expression on IMiD-insensitive MM cells and plasma cells from patients newly diagnosed with MM. Gene-expression profiling analysis demonstrated that not only PD-L1, but also a proliferation-inducing ligand (APRIL), was enhanced by IMiDs. PD-L1 induction by IMiDs was suppressed by using the APRIL inhibitor recombinant B-cell maturation antigen (BCMA)-Ig, the antibody against BCMA, or an MEK/ERK inhibitor in in vitro and in vivo assays. In addition, its induction was abrogated in cereblon (CRBN)-knockdown MM cells, whereas PD-L1 expression was increased and strongly induced by IMiDs in Ikaros-knockdown cells. These results demonstrated that PD-L1 upregulation by IMiDs on IMiD-insensitive MM cells was induced by (i) the BCMA-APRIL pathway via IMiD-mediated induction of APRIL and (ii) Ikaros degradation mediated by CRBN, which plays a role in inhibiting PD-L1 expression. Furthermore, T-cell inhibition induced by PD-L1-upregulated cells was effectively recovered after combination treatment with durvalumab and IMiDs. PD-L1 upregulation by IMiDs on MM cells might promote aggressive myeloma behaviors and immune escape in the bone marrow microenvironment.

Circulating Cancer Cells with Relative Expression of miRNA and Cytotoxic T-cell Activities in Non-small cell Lung Cancer Patients: PhD Thesis Abstract

Background: Lung cancer is the leading cause of cancer-related death worldwide with a substantially low survival rate. MiRNAs have been shown to regulate self-renewal and differentiation properties of CSCs, Here, MicroRNAs play a significant role in shaping immune response. Aims: the current study aims to analyze the numbers of helper CD4+ and cytotoxic CD8+ T lymphocytes, CD133+ and Epcam+ CSCs. also, measure the relative expression of miRNAs in Non-small cell lung cancer patients before and after treatment. Materials and Methods: The frequencies of Epcam+ and CD133+ cells and the numbers of helper CD4+ and cytotoxic CD8+ T lymphocytes were analyzed in the peripheral blood of NSCLC patients before (n= 10) and after (n= 10) chemotherapy using multiparametric flow cytometry. The gene expression of miRNAs was measured using microarray.Results: Early diagnosed patients before treatment showed high numbers of Epcam+ and CD113+ CSCs when compared to CTRL; where the numbers of these cells were decreased after treatment as compared to early diagnosed patients. A significant upregulation of miRNAs and downregulation of miRNAs were observed in the peripheral blood of NSCLC patients as compared to healthy control. The relative and absolute numbers of CD4+ and CD8+ T-cells were significantly elevated in the peripheral blood of NSCLC patients. Conclusion. This study opens a new avenue to investigate the mechanism mediating the emergence of these cells on larger number of NSCLC patients at different treatment stages. We also discuss the role of microRNAs in therapeutic resistance and as biomarkers.

Growth Hormone Releasing Hormone Reduces Circulating Markers of Immune Activation in Parallel with Effects on Hepatic Immune Pathways in Individuals with HIV-infection and Nonalcoholic Fatty Liver Disease.

The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis modulates critical metabolic pathways; however, little is known regarding effects of augmenting pulsatile GH secretion on immune function in humans. This study used proteomics and gene set enrichment analysis to assess effects of a GH releasing hormone (GHRH) analog, tesamorelin, on circulating immune markers and liver tissue in people with human immunodeficiency virus (HIV) (PWH) and nonalcoholic fatty liver disease (NAFLD). 92 biomarkers associated with immunity, chemotaxis, and metabolism were measured in plasma samples from 61 PWH with NAFLD who participated in a double-blind, randomized trial of tesamorelin versus placebo for 12 months. Gene set enrichment analysis was performed on serial liver biopsies targeted to immune pathways. Tesamorelin, compared to placebo, decreased interconnected proteins related to cytotoxic T-cell and monocyte activation. Circulating concentrations of 13 proteins were significantly decreased, and no proteins increased, by tesamorelin. These included 4 chemokines (CCL3, CCL4, CCL13 [MCP4], IL8 [CXCL8]), 2 cytokines (IL-10 and CSF-1), and 4 T-cell associated molecules (CD8A, CRTAM, GZMA, ADGRG1), as well as ARG1, Gal-9, and HGF. Network analysis indicated close interaction among the gene pathways responsible for these proteins, with imputational analyses suggesting down-regulation of a closely related cluster of immune pathways. Targeted transcriptomics using liver tissue confirmed a significant end-organ signal of down-regulated immune activation pathways. Long-term treatment with a GHRH analog reduced markers of T-cell and monocyte/macrophage activity, suggesting that augmentation of the GH axis may ameliorate immune activation in an HIV population with metabolic dysregulation, systemic and end organ inflammation. Clinical Trials Registration. NCT02196831.

Immune modulating activity of the CHK1 inhibitor prexasertib and anti-PD-L1 antibody LY3300054 in patients with high-grade serous ovarian cancer and other solid tumors.

Checkpoint kinase 1 (CHK1) has dual roles in both the DNA damage response and in the innate immune response to genotoxic stress. The combination of CHK1 inhibition and immune checkpoint blockade has the potential to enhance anti-tumoral T-cell activation. This was an open-label phase 1 study evaluating the CHK1 inhibitor prexasertib and the anti-PD-L1 antibody LY3300054. After a lead-in of LY3300054 (Arm A), prexasertib (Arm B) or the combination (Arm C), both agents were administered intravenously at their respective recommended phase 2 doses (RP2Ds) on days 1 and 15 of a 28-day cycle. Flow cytometry of peripheral blood was performed before and during treatment to analyze effects on immune cell populations, with a focus on T cell subsets and activation. Plasma cytokines and chemokines were analyzed using the Luminex platform. Among seventeen patients enrolled, the combination was tolerable at the monotherapy RP2Ds, 105mg/m2 prexasertib and 700mg LY3300054. Dose-limiting toxicities included one episode each of febrile neutropenia (Arm C) and grade 4 neutropenia lasting > 5days (Arm B). One patient had immune-related AST/ALT elevation after 12 cycles. Three patients with CCNE1-amplified, high-grade serous ovarian cancer (HGSOC) achieved partial response (PR), 2 lasting > 12months; a fourth such patient maintained stable disease > 12months. Analysis of peripheral blood demonstrated evidence of CD8 + T-cell activation in response to treatment. Prexasertib in combination with PD-L1 blockade was tolerable and demonstrated preliminary activity in CCNE1-amplified HGSOC with evidence of cytotoxic T-cell activation in patient blood samples. ClinicalTrials.gov identifier: NCT03495323. Registered April 12, 2018.

P78.11 Immunotherapy-Induced Coeliac Disease in the Curative Lung Cancer Patient on Adjuvant Durvalumab

The PACIFIC study led to the approval of durvalumab in the adjuvant setting for patients with unresectable, stage III non-small cell lung cancer (NSCLC) after chemo-radiotherapy (CRT)1, and whose tumours express programmed death-ligand-1 (PDL-1) in >1% of cells. PDL-1 is an immune regulating molecule which has been hypothesized to aid neoplastic lesions to evade the host’s immune system. Durvalumab is a human monoclonal IgG1 antibody which blocks PDL-1 on cytotoxic T-cells, leading to increased cytotoxic T-cell proliferation and activity, and cytokine production1. The activation of T-cells can lead to immune-related adverse events (IrAEs). A 68-year-old female with stage III NSCLC received adjuvant durvalumab. Her PDL-1 was 90-100%. After four cycles of durvalumab, she presented with Grade 2 diarrhoea2. She was admitted to hospital and started on prednisone 50mg (1mg/kg). After three days, she had a sigmoidoscopy which appeared macroscopically normal. Biopsies showed a non-specific increase in intraepithelial lymphocytes. The diarrhoea resolved, and she was discharged on a weaning regime of prednisolone for presumed immune checkpoint inhibitor (ICPi)-induced colitis. Two months later, she represented with Grade 3 diarrhea2. Examination and imaging was normal. Positive findings were an elevated serum anti-transglutaminase (anti-tTG) IgA level at 10.2unit/mL, low folic acid and low vitamin D. An oesophagogastroduodenoscopy (OGD) showed scalloping of the duodenum and biopsies showed marked villous blunting, chronic inflammation of the lamina propria and increased intraepithelial lymphocytes; all findings consistent with the diagnosis of CD. She was started on a lifelong gluten-free (GF) diet and given a weaning course of oral prednisone with the intention of improving her symptoms rapidly. Her diarrhoea immediately improved and a decision was made to restart immunotherapy once the steroids were complete. She did not report recurrence of her symptoms following an additional seven cycles of durvalumab therapy whilst on a GF diet. The anti-tTG IgA normalised after 3 months. This case highlights a rare IrAE of CD presenting during ICPi treatment. Durvalumab may have unmasked previously undiagnosed CD. Alternatively, the patient may have developed CD de-novo, as a direct consequence of ICPi treatment. The detection of rare toxicities requires a large number of patients to be treated with ICPis, therefore rarer toxicities are often identified post licensing, when the number of patients receiving these drugs increases exponentially. There are three published cases of patients receiving immunotherapy developing CD after the commencement of the drug but the exact incidence of this IrAE and exact underlying mechanism for the development of CD secondary to immunotherapy is unknown. This is the first reported case of CD arising following ICPis in both the adjuvant lung cancer setting, and with the drug durvalumab. A new diagnosis of CD will have long-term consequences for this patient who may have been cured with CRT alone. However, the majority of patients with stage III NSCLC have disease progression despite CRT and therefore the addition of immunotherapy in improving survival is significant. Although diarrhoea due to CD is an exceptional IrAE, symptoms can resolve quickly once a diagnosis is made and a GF diet commenced.

Phase II open-label study of S-588410 as maintenance monotherapy after first-line platinum-containing chemotherapy in patients with advanced or metastatic urothelial carcinoma.

440 Background: S-588410 is a cancer peptide vaccine composed of 5 human leukocyte antigen (HLA)-A*24:02-restricted epitope peptides derived from 5 cancer-testis antigens: DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1; all of which are highly expressed in urothelial carcinoma. This study aimed to evaluate the effect of S-588410 maintenance therapy on peptide-specific cytotoxic T-lymphocyte (CTL) induction in patients with advanced or metastatic urothelial carcinoma after first-line platinum-based chemotherapy. Methods: An open-label, multicenter phase II trial was performed across 62 sites in Japan, the United Kingdom, France and Bulgaria (EudraCT 2013-005274-22). Eligible patients had completed ≥4 cycles of first-line platinum-based chemotherapy without disease progression. HLA-A*24:02-positive patients received S-588410 (1 mg of each of 5 peptides mixed with Montanide ISA 51 VG) subcutaneously weekly for 12 weeks, then every 2 weeks for up to 2 years. HLA-A*24:02-negative patients were enrolled in an observation group and did not receive study drug. The primary endpoint for the S-588410 group was the CTL induction rate at 12 weeks, defined as the proportion of patients who showed increased CTL activity for ≥1 peptide. Secondary endpoints included CTL induction rate after 1 year, antitumor effect defined by immune-related response criteria, progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 81 patients with platinum-sensitive advanced or metastatic urothelial carcinoma were enrolled (S-588410 group, n=45; observation group, n=36) between April 2014 and November 2017. Most patients were male and Asian with a mean age of 67 years. CTLs were induced in 42 (93.3%) patients who received S-588410 for 12 weeks (P<0.0001, one-sided binomial test where the CTL induction rate is ≤50% as the null hypothesis). The CTL induction rate steadily increased to 95.6% within 48 weeks. CTL activity was high for the DEPDC1, MPHOSPH1 and URLC10 peptides. The response rate (immune-related complete response [CR] or partial response [PR]) was 8.9% (4/45 patients) in the S-588410 group and 0% in the observation group. Tumor imaging showed gradual (PR, n=3) and durable (CR, n=1) tumor shrinkage after ≥36 weeks in the S-588410 group. Median PFS was 18.1 weeks in the S-588410 group and 12.5 weeks in the observation group. Median OS was 71 and 99 weeks, respectively. The most frequent treatment-emergent adverse event was injection site reaction (42/45 patients [93.3%]; Grades 1–3). Pyrexia, rash and pruritus were also observed in the S-588410 group, but not the observation group. Conclusions: S-588410 showed a potent immune response and acceptable safety profile in patients with advanced or metastatic urothelial carcinoma, potentially offering a clinical benefit as post-chemotherapy maintenance therapy. Clinical trial information: EudraCT 2013-005274-22.

Chimeric Virus-Like Particles of Prawn Nodavirus Displaying Hepatitis B Virus Immunodominant Region: Biophysical Properties and Cytokine Response.

Hepatitis B is a major global health challenge. In the absence of an effective treatment for the disease, hepatitis B vaccines provide protection against the viral infection. However, some individuals do not have positive immune responses after being vaccinated with the hepatitis B vaccines available in the market. Thus, it is important to develop a more protective vaccine. Previously, we showed that hepatitis B virus (HBV) ‘a’ determinant (aD) displayed on the prawn nodavirus capsid (Nc) and expressed in Spodoptera frugiperda (Sf9) cells (namely, Nc-aD-Sf9) self-assembled into virus-like particles (VLPs). Immunisation of BALB/c mice with the Nc-aD-Sf9 VLPs showed significant induction of humoral, cellular and memory B-cell immunity. In the present study, the biophysical properties of the Nc-aD-Sf9 VLPs were studied using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was used to determine the antigenicity of the Nc-aD-Sf9 VLPs, and multiplex ELISA was employed to quantify the cytokine response induced by the VLPs administered intramuscularly into BALB/c mice (n = 8). CD spectroscopy of Nc-aD-Sf9 VLPs showed that the secondary structure of the VLPs predominantly consisted of beta (β)-sheets (44.8%), and they were thermally stable up to ~52 °C. ELISA revealed that the aD epitope of the VLPs was significantly antigenic to anti-HBV surface antigen (HBsAg) antibodies. In addition, multiplex ELISA of serum samples from the vaccinated mice showed a significant induction (p < 0.001) of IFN-γ, IL-4, IL-5, IL-6, IL-10, and IL-12p70. This cytokine profile is indicative of natural killer cell, macrophage, dendritic cell and cytotoxic T-lymphocyte activities, which suggests a prophylactic innate and adaptive cellular immune response mediated by Nc-aD-Sf9 VLPs. Interestingly, Nc-aD-Sf9 induced a more robust release of the aforementioned cytokines than that of Nc-aD VLPs produced in Escherichia coli and a commercially used hepatitis B vaccine. Overall, Nc-aD-Sf9 VLPs are thermally stable and significantly antigenic, demonstrating their potential as an HBV vaccine candidate.

Targeting MARCO and IL37R on Immunosuppressive Macrophages in Lung Cancer Blocks Regulatory T Cells and Supports Cytotoxic Lymphocyte Function.

The progression and metastatic capacity of solid tumors are strongly influenced by immune cells in the tumor microenvironment. In non-small cell lung cancer (NSCLC), accumulation of anti-inflammatory tumor-associated macrophages (TAM) is associated with worse clinical outcome and resistance to therapy. Here we investigated the immune landscape of NSCLC in the presence of protumoral TAMs expressing the macrophage receptor with collagenous structure (MARCO). MARCO-expressing TAM numbers correlated with increased occurrence of regulatory T cells and effector T cells and decreased natural killer (NK) cells in these tumors. Furthermore, transcriptomic data from the tumors uncovered a correlation between MARCO expression and the anti-inflammatory cytokine IL37. In vitro studies subsequently showed that lung cancer cells polarized macrophages to express MARCO and gain an immune-suppressive phenotype through the release of IL37. MARCO-expressing TAMs blocked cytotoxic T-cell and NK-cell activation, inhibiting their proliferation, cytokine production, and tumor killing capacity. Mechanistically, MARCO+ macrophages enhanced regulatory T (Treg) cell proliferation and IL10 production and diminished CD8 T-cell activities. Targeting MARCO or IL37 receptor (IL37R) by antibody or CRISPR knockout of IL37 in lung cancer cell lines repolarized TAMs, resulting in recovered cytolytic activity and antitumoral capacity of NK cells and T cells and downmodulated Treg cell activities. In summary, our data demonstrate a novel immune therapeutic approach targeting human TAMs immune suppression of NK- and T-cell antitumor activities. SIGNIFICANCE: This study defines tumor-derived IL37 and the macrophage scavenger receptor MARCO as potential therapeutic targets to remodel the immune-suppressive microenvironment in patients with lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/4/956/F1.large.jpg.

Activatable Polymeric Nanoprobe for Near‐Infrared Fluorescence and Photoacoustic Imaging of T Lymphocytes

AbstractDevelopment of real‐time non‐invasive imaging probes to assess infiltration and activation of cytotoxic T cells (CTLs) is critical to predict the efficacy of cancer immunotherapy, which however remains challenging. Reported here is an activatable semiconducting polymer nanoprobe (SPNP) for near‐infrared fluorescence (NIRF) and photoacoustic (PA) imaging of a biomarker (granzyme B) associated with activation of CTLs. SPNP comprises a semiconducting polymer (SP) conjugated with a granzyme B cleavable and dye‐labeled peptide as the side chain, both of which emit NIRF and PA signals. After systemic administration, SPNP passively targets the tumor and in situ reacts with granzyme B to release the dye‐labeled peptide, leading to decreased NIRF and PA signals from the dye but unchanged signals from the polymer. Such ratiometric NIRF and PA signals of SPNP correlate well with the expression level of granzyme B and intratumoral population of CTLs. Thus, this study not only presents the first PA probes for in vivo imaging of immune activation but also provides a molecular design strategy that can be generalized for molecular imaging of other immune‐related biomarkers.

Activatable Polymeric Nanoprobe for Near-Infrared Fluorescence and Photoacoustic Imaging of T Lymphocytes.

Development of real-time non-invasive imaging probes to assess infiltration and activation of cytotoxic T cells (CTLs) is critical to predict the efficacy of cancer immunotherapy, which however remains challenging. Reported here is an activatable semiconducting polymer nanoprobe (SPNP) for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of a biomarker (granzyme B) associated with activation of CTLs. SPNP comprises a semiconducting polymer (SP) conjugated with a granzyme B cleavable and dye-labeled peptide as the side chain, both of which emit NIRF and PA signals. After systemic administration, SPNP passively targets the tumor and in situ reacts with granzyme B to release the dye-labeled peptide, leading to decreased NIRF and PA signals from the dye but unchanged signals from the polymer. Such ratiometric NIRF and PA signals of SPNP correlate well with the expression level of granzyme B and intratumoral population of CTLs. Thus, this study not only presents the first PA probes for in vivo imaging of immune activation but also provides a molecular design strategy that can be generalized for molecular imaging of other immune-related biomarkers.

Abstract PO083: Treatment of CEA-positive solid tumors with anti-CEA chimeric antigen receptor T-cells in CEA transgenic mice

Abstract Chimeric antigen receptors (CARs) combine specificity of antibody to target antigen on cancer cells with the potent cytotoxic activity of T-cells without the need for MHC recognition. Chimeric antigen receptor (CAR) T-cells that were designed to express the single chain variable fragment binding domain of human anti-CEA antibody BW431/26 on mouse splenocytes were able to reduce CEA-expressing pancreatic adenocarcinoma tumor in CEA-transgenic (CEAtg) mice without significant off-target effects (Chmielewski et al., 2012). In this study, an anti-CEA CAR construct derived from murine anti-CEA antibody (T84.66) expressed on human T-cells targeted CEA+ colon carcinoma cells (LS174T/CEA and MC38CEA) with high specificity in vitro. When co-incubated with CEA-positive or CEA-negative mouse cancer cells, anti-CEA CAR expressed on mouse T-cells also specifically targeted CEA-positive cancer cells in vitro. Compared to mock T-cells control, anti-CEA CAR T-cells injected intravenously delayed subcutaneous MC38CEA tumor growth in CEA-transgenic mice (CEAtg). Lymphodepletion via cyclophosphamide injected before anti-CEA T-cells injection further delayed subcutaneous MC38CEA tumor growth and delayed orthotopic CEA+ breast carcinoma (E0771CEA) tumor growth in CEAtg mice. To improve the therapeutic potential of anti-CEA CAR T-cells, IL2 conjugated to humanized anti-CEA antibody (M5A-IL2, ICK) was interperitoneally injected after anti-CEA CAR T-cells into lymphodepleted CEAtg mice. A single injection of ICK after anti-CEA CAR T-cells delayed subcutaneous MC38CEA tumor growth even further. Two injections of ICK after anti-CEA T-cells delayed orthotopic E0771CEA tumor growth. Four injections of ICK after anti-CEA CAR T-cells eradicated subcutaneous MC38CEA tumors. These data show the therapeutic potential of anti-CEA CAR T-cells to target CEA-positive tumors. Citation Format: Seung Cha, Paul Yazaki, Christine Brown, John Shively. Treatment of CEA-positive solid tumors with anti-CEA chimeric antigen receptor T-cells in CEA transgenic mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO083.

A Hepatitis B Virus-Derived Peptide Exerts an Anticancer Effect via TNF/iNOS-producing Dendritic Cells in Tumor-Bearing Mouse Model.

Simple SummaryRecently, it has been reported that a tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cell (Tip-DC) may play a pivotal role in the anticancer immune response by activating CD8+ T cells in tumor microenvironments. The development of a new immunotherapeutic agent that can enhance the oncolytic effect of Tip-DC has gained increasing attention in the cancer research field. In this study, we introduce a hepatitis B virus-derived peptide, Poly6, which elicited a strong anticancer immune response via enhanced Tip-DC activity. Our findings suggest that Poly6 could be a novel potential adjuvant/co-treatment partner in anticancer immunotherapy approaches.Recently, we reported a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, that exerts antiviral effects against human immunodeficiency virus type 1 (HIV-1). Here, we explored the immunotherapeutic potential of Poly6 via its administration into dendritic cells (DCs) in a mouse model. Our data revealed that Poly6 treatment led to enhanced production of tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner via the induction of mitochondrial stress. Poly6 treatment in mice implanted with MC38 cells, a murine colon adenocarcinoma line, led to attenuated tumor formation, primarily due to direct cell death induced by Tip-DC mediated nitric oxide (NO) production and indirect killing by Tip-DC mediated cluster of differentiation 8 (CD8) cytotoxic T lymphocyte (CTL) activation via CD40 activation. Moreover, Poly6 treatment demonstrated an enhanced anticancer effect with one of the checkpoint inhibitors, the anti PD-L1 antibody. In conclusion, our data reveal that Poly6 treatment elicits an antitumor immune response in mice, possibly through NO-mediated oncolytic activity via Tip-DC activation and Tip-DC mediated CTL activation. This suggests that Poly6 represents a potential adjuvant for cancer immunotherapy by enhancing the anticancer effects of immune checkpoint inhibitors.

A Systematic Review of Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia: Engaging an Old Problem With New Solutions.

To assess the current literature for blinatumomab in the treatment of adult and pediatric B-cell acute lymphoblastic leukemia (ALL). We conducted a PubMed (inception to December 11, 2020) and ClinicalTrials.gov systematic literature search using the following terms: blinatumomab, Blincyto, lymphoblastic leukemia, and bispecific T-cell engager. All relevant published articles, package inserts, and meeting abstracts evaluating the use of blinatumomab in ALL were considered for inclusion. Blinatumomab, a first-in-class bispecific T-cell engager monoclonal antibody, facilitates cytotoxic T-cell activation and subsequent eradication of CD19-positive B cells. The confirmatory phase III TOWER trial demonstrated superior overall survival (OS) with blinatumomab compared with standard chemotherapy (7.7 months vs 4.0 months) in relapsed and refractory (R/R) B-cell ALL. In the phase II BLAST trial, blinatumomab achieved a complete measurable residual disease (MRD) response in 78% of evaluable patients, with a median OS of 36.5 months. Potentially life-threatening cytokine release syndrome and neurotoxicity occurred in approximately 15% and 65% of patients, respectively. Following initial Food and Drug Administration approval in 2014, blinatumomab gained expanded approval in pediatric patients and in Philadelphia chromosome-positive R/R ALL. In 2018, blinatumomab became the first and only drug approved for the treatment of persistent MRD in any hematologic malignancy. Emerging data demonstrate promising efficacy with blinatumomab in specific ALL settings, including frontline therapy, as a bridge to transplantation, and in "chemotherapy-free" combination regimens. Blinatumomab provides a paradigm-shifting treatment option; however, many questions surrounding optimal patient selection, sequencing, and cost-effectiveness remain.

RETRACTED ARTICLE: A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents.

Application of a newly-developed cynomolgus macaque BiTE-mediated cytotoxic T-lymphocyte activity assay to various immunomodulatory agents in vitro

The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of in vitro and in vivo immunophenotyping and functional study endpoints in pre-clinical studies. CD8+ cytotoxic T-lymphocyte (CTL) activity impairment by immunosuppressive agents is recognized to be a potentiating factor for decreased antiviral defense and increased cancer risk. A bi-specific T-cell engager (BiTE®)-mediated CTL activity assay that applies to ex vivo experimentation in non-human primates in the context of toxicology studies was successfully developed and applied in cynomolgus monkey regulatory studies. While an ex vivo analysis conducted in the context of repeat-dose toxicology studies focuses on the long-term impact on CTL function, an in vitro assay with the same experimental design captures acute effects in the presence of the test article. Here, the in vitro assay was applied to a list of drugs with known clinical immunomodulatory impact to understand the applicability of the assay. The results showed this assay was sensitive to a wide range of immunosuppressants directly targeting cell-intrinsic signaling pathways in activated CTL. However, agents executing immuno-modulation through inhibiting cytokines/cytokine receptors, co-stimulatory molecules, and cell adhesion and migration pathways did not impair the CTL activity in this short-term in vitro culture. In addition, anti-PD-1/PD-L1 immune checkpoint blockers enhanced the CTL activity. Taken together, the results here demonstrate that in concordance with their mechanism of action, the in vitro BiTE®-mediated CTL assay is applicable and sensitive to immunomodulatory agents acting via a variety of mechanisms.

CTL ELISPOT Assay and T Cell Detection.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

Immunotoxicity studies of sulfolane following developmental exposure in Hsd:Sprague Dawley SD rats and adult exposure in B6C3F1/N mice

Sulfolane is a solvent used in the petrochemical industry and a groundwater contaminant in areas near refineries. The current studies were conducted to assess the impact of oral exposure to sulfolane on the immune system using two models: (1) a perinatal drinking water exposure to 0, 30, 100, 300, or 1000 mg/L from gestation day (GD) 6 until ∼13 weeks-of-age in Harlan Sprague Dawley rats; and, (2) a 90-day gavage exposure of adult female B6C3F1/N mice to 0, 1, 10, 30, 100, or 300 mg/kg/day. Immune parameters evaluated included measurement of antibody production against sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), ex vivo measurements of natural killer (NK) cell activity, cytotoxic T-cell (CTL) activity, and T-cell proliferation, as well as measures of splenic immune cell populations, hematological parameters, and histopathology of immune tissues. A decrease in ex vivo NK cell activity was observed in cells from female – but not male – F1 rats following developmental exposure. In adult female mice, splenic NK cell number was lower than the vehicle controls at doses ≥ 100 mg/kg; however, ex vivo NK cell activity was not affected by sulfolane treatment. In female mice, a decrease in the number of large unstained cells at doses ≥ 30 mg/kg was observed. In F1 rats, effects on white blood cells (WBC) were limited to a decreasing trend in leukocytes in females; no effects were observed in males. Under the conditions of this study, a no-observed-effect level (NOEL) of 3 mg/kg/day was identified based on reduced NK cell activity in female F1 rats. Overall, these findings suggest that oral exposure to sulfolane in rodents had minimal effects on the immune system.

Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma.

Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

Гемофагоцитарный синдром: механизмы развития, клинические проявления, терапевтические технологии

Hemophagocytic lymphohistiocytosis (HLH), also known as cytokine storm, hemophagocytic lymphohistiocytosis, and macrophage activation syndrome, is a life-threatening disorder caused by a congenital or acquired defect in the cytolytic activity of cytotoxic T-lymphocytes (CD8+) and natural killers, as well as hyperproduction of interleukins-1, -6, -18, and interferon-γ. HLH develops in children with systemic juvenile idiopathic arthritis (sJIA), often resulting in multiple organ failure, and is characterized by a high risk of death. This article discusses the mechanisms underlying the development of primary and secondary HLH and sJIA, as well as clinical and laboratory manifestations of HLH. It summarizes the information on diagnostic criteria and analyzes therapeutic approaches used for HLH. Key words: hemophagocytic lymphohistiocytosis, systemic juvenile idiopathic arthritis, biologicals, rheumatology, children