Cytotoxic T-lymphocyte Responses Research Articles

Annona muricata L.-Derived Polysaccharides as a Potential Adjuvant to a Dendritic Cell-Based Vaccine in a Thymoma-Bearing Model.

Dendritic cells (DCs) are powerful antigen-presenting cells that are often used to evaluate adjuvants, particularly for adjuvant selection for various vaccines. Here, polysaccharides (named ALP) isolated from leaves of Annona muricata L., which are used in traditional medicine such as for bacterial infections and inflammatory diseases, were evaluated as an adjuvant candidate that can induce anti-tumor activity. We first confirmed the phenotypic (surface molecules, cytokines, antigen uptake, and antigen-presenting ability) and functional alterations (T cell proliferation/activation) of DCs in vitro. We also confirmed the adjuvant effect by evaluating anti-tumor activity and immunity using an ALP-treated DC-immunized mouse model. ALP functionally induced DC maturation by up-regulating the secretion of Th1-polarizing pro-inflammatory cytokines, the expression of surface molecules, and antigen-presenting ability. ALP triggered DC maturation, which is dependent on the activation of the MAPK and NF-κB signaling pathways. ALP-activated DCs showed an ample capacity to differentiate naive T cells to Th1 and activated CD8+ T cells effectively. The systemic administration of DCs that pulse ALP and ovalbumin peptides strongly increased cytotoxic T lymphocyte (CTL) activity (by 9.5% compared to that in the control vaccine groups), the generation of CD107a-producing multifunctional T cells, and Th1-mediated humoral immunity, and caused a significant reduction (increased protection by 29% over that in control vaccine groups) in tumor growth. ALP, which triggers the Th1 and CTL response, provides a basis for a new adjuvant for various vaccines.

Cytosolic delivery of HBsAg and enhanced cellular immunity by pH-responsive liposome

It remains a major challenge to prime the cytotoxic T lymphocytes (CTLs) response for the treatment of Hepatitis B virus (HBV) infection. Inspired by an important natural biological behavior, membrane fusion, we constructed a pH-responsive nanocarrier (HBsAg&CpG@Lip) with a membrane fusion capacity for HBsAg intracellular delivery and subsequent process of CTLs. In the in vitro experiments, HBsAg&CpG@Lip greatly promoted the cellular uptake of HBsAg and the activation of BMDCs. It induced the cytosolic release of HBsAg in BMDCs, which was in conformity with the membrane fusion capacity of HBsAg&CpG@Lip. Such a capacity was improved by 4.4-fold in pH = 5.0 than that in pH = 7.4 at 60 min, which was calculated through fluorescence resonance energy transfer (FRET) efficiency. Furthermore, the cross-presentation activity induced by liposome at 1 μg/mL was equivalent to that by soluble antigen at 2000 μg/mL, which indicated an advantage for the liposome in cytosolic antigen delivery and potent CTLs activation. In respect of the transport of liposomes into draining lymph nodes (DLNs), the recruitment of liposome+migratory DCs at 24 h raised 11-fold than that at 3 h, suggesting that liposomes were mainly carried by migratory DCs. Meanwhile, HBsAg&CpG@Lip also elicited the activation of DCs and the generation of Tfh cells in DLNs. After immunization, HBsAg&CpG@Lip induced a higher anti-HBsAg IgG and ratio of IgG2c/IgG1 than that by HBsAg or Alum+HBsAg group. It also augmented a strong CTLs response as expected. Specifically, stimulation with HBsAg&CpG@Lip increased the number of IFN-γ-secreting splenocytes, the proportion of CD107α+CD8+and FasL+CD8+ T cells and the secretion of Granzyme B, which verified the design of membrane fusion in vivo. In summary, this design for HBsAg cytosolic delivery exhibits great benefits for triggering CTLs, opening an avenue for the development of a potent therapeutic HBV vaccine.

Recombinant PAL/PilE/FlaA DNA vaccine provides protective immunity against Legionella pneumophila in BALB/c mice

BackgroundLegionella pneumophila (L.pneumophila), a Gram-negative small microorganism, causes hospital-acquired pneumonia especially in immunocompromised patients. Vaccination may be an effective method for preventing L.pneumophila infection. Therefore, it is necessary to develop a better vaccine against this disease. In this study, we developed a recombinant peptidoglycan-associated lipoprotein (PAL)/type IV pilin (PilE)/lagellin (FlaA) DNA vaccine and evaluated its immunogenicity and efficacy to protect against L.pneumophila infection.ResultsAccording to the results, the expression of PAL, PilE, FlaA proteins and PAL/PilE/FlaA fusion protein in 293 cells was confirmed. Immunization with PAL/PilE/FlaA DNA vaccine resulted in highest IgG titer and strongest cytotoxic T-lymphocyte (CTL) response. Furthermore, the histopathological changes in lung tissues of mice challenged with a lethal dose of L.pneumophila were alleviated by PAL/PilE/FlaA DNA vaccine immunization. The production of T-helper-1 (Th1) cytokines (IFNγ, TGF-α, and IL-12), and Th2 cytokines (IL-4 and IL-10) were promoted in PAL/PilE/FlaA DNA vaccine group. Finally, immunization with PAL/PilE/FlaA vaccine raised the survival rate of mice to 100% after challenging with a lethal dose of L.pneumophila for 10 consecutive days.ConclusionsOur study suggests that the newly developed PAL/PilE/FlaA DNA vaccine stimulates strong humoral and cellular immune responses and may be a potential intervention on L.pneumophila infection.

A longitudinal analysis of TCRβ repertoire diversity in immune tolerance of pediatric patients post-UCBT: T cell clonal deletion over clonal anergy

Background & Aim The mechanisms of long-term immune tolerance after HLA mismatched unrelated cord blood transplant (UCBT) is not understood. Both central (clonal deletion) and peripheral mechanisms (anergy, Treg, or Tr1 mediated suppression) are suspected. In this study, we set up serial experiments to test for each of these mechanisms in the GvH direction. Next-generation TCR sequencing was applied for tracking and quantitating alloreactive T cell clones within the TCRVβ repertoire. Methods, Results & Conclusion Methods: 12 patients (median age 1y/o) with non-malignant diseases were enrolled on a reduced-intensity conditioning trial (NCT01852370) with Alemtuzumab 2 weeks pre-UCBT. Patients were tolerant if completed immunocompetent, off immunosuppression for months without GvHD or rejection. T cell responses to host antigen-presenting cells (APC) were measured utilizing EBV transformed B cells in seronegative children by mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) reaction, along with cytokine profiling and ImmunoSEQ® monitoring the fate of host-reactive T cell clones. To identify the key mechanisms of tolerance, we depleted Tregs with Denileukin diftitox prior to MLR and CTL. The involvement of Tr1 or anergy was examined either by blocking IL10R or by adding low dose IL2, respectively. Result and discussion In tolerant patients, there was no significant proliferative or CTL response towards host APC by donor-derived T cells while they responded vigorously to 3rd party APC (Fig1A). Similarly, cytokine measurements by Bioplex revealed host-specific non-responsiveness (Fig1B) similar to anti-self-responses of healthy volunteers. TCRβ clonotype tracking with ImmunoSEQ® revealed the disappearance of host-specific clones identifiable from an aliquot of the infused CB graft (pre-UCBT) (Fig1C). These alloreactive T cell clones became undetectable even in the presence of exogenous IL2 (Fig1D). There was no indication of Treg or Tr1 involvement in sustaining tolerance since neither deletion of Tregs, nor IL-10R blockade could reverse T cell hypo-reactivity in MLR, CTL, and Bioplex assays. In summary, the rapid acquisition of immune tolerance in the GvH direction post-UCBT is characterized by hypo-reactivity towards host APC in proliferation, cytotoxicity, cytokine secretion. While a small degree of anergy may also contribute, ImmunoSEQ® data confirmed that clonal deletion is a dominant mechanism towards long term immune tolerance.

Endogenous TLR2 ligand embedded in the catalytic region of human cysteinyl-tRNA synthetase 1

BackgroundThe generation of antigen-specific cytotoxic T lymphocyte (CTL) responses is required for successful cancer vaccine therapy. In this regard, ligands of Toll-like receptors (TLRs) have been suggested to activate adaptive...

Mechanistic mathematical representation of CD8+ T cell mediated drug-induced liver injury (DILI) Part 2: translation from experimental antigen, ovalbumin (OVA), to drug, amodiaquine (AQ) response

Abstract Idiosyncratic DILI (iDILI) is poorly understood and costly to patients and drug developers. Some iDILI events appear immune-mediated. This work builds on the DILIsym representation of T cell cytotoxicity against OVA-expressing hepatocytes1, to translate the OVA representation to AQ-induced hepatotoxicity. AQ-related changes include a physiologically-based pharmacokinetic (PBPK) model for mouse AQ exposure, AQ metabolism to reactive metabolites (RMs), RM-mediated ER stress leading to inflammation, fewer endogenous AQ responsive T cells of reduced affinity and avidity for AQ antigen. Simulations show no CD8+ T cell mediated ALT elevations in simulated wild-type mice treated with AQ, consistent with data2 and provide a mechanistic basis for the result. With low levels of AQ antigen, low T cell numbers and lower affinity and avidity interactions were insufficient for a cytotoxic T lymphocyte (CTL) response. With high levels of AQ antigen, T cell expansion and differentiation occurred but regulatory pathways, i.e., T cell exhaustion, inhibited an effective CTL response. In PD1−/− mice treated with anti-CTLA4, CD8+ T cell mediated AQ DILI was apparent (ALT >100 U/L)3,4. In DILIsym, blocking coinhibitory activity on proliferation and differentiation was permissive for AQ DILI (ALT >100 U/L) and could be further exacerbated by blocking differentiation to an exhausted phenotype (ALT >200 U/L). The results provide proof-of-concept for translation of OVA hepatotoxicity to AQ DILI and set the stage for exploration of human AQ DILI5.

Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy

Abstract In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Dendritic Cell-Based Therapy Using LY6E Peptide with a Putative Role Against Colorectal Cancer.

IntroductionAlbeit early stage gastrointestinal (GI) carcinomas have a good prognosis if treated with surgery, diagnosis is often confirmed at a late stage and efficacious drugs are lacking. Recent progress in immune-based therapies has focused on dendritic cells (DCs), aiming to elicit tumor-specific responses by inducing immunological memory. Our previous microarray study indicated that a biomarker, termed lymphocyte antigen-6E (LY6E), is commonly overexpressed in two potentially lethal GI cancers: those of colon and stomach. In this study, we examined the antigenic potency of LY6E in stimulating DCs.MethodsFollowing isolation, differentiation, and maturation of mononuclear cells, DCs were pulsed with LY6E peptide, a protein related to major histocompatibility complex (MHC) class I/II. Subsequently, DCs were co-cultured with mouse splenocytes to assess antigen-specific T-cell proliferation. Elucidated cytotoxic T-lymphocyte responses were assessed using subcutaneous colorectal murine tumor models.ResultsOur in vitro results suggest that DCs loaded with LY6E peptide antigen are capable of stimulating and inducing proliferation of murine T-cells. Furthermore, our in vivo results demonstrate that LY6E peptide has a substantial impact on provoking immune responses against induced colon cancer in mice.DiscussionIn conclusion, based on the overexpression of LY6E in colorectal, gastric, and pancreatic cancers, the role of this peptide should be further investigated with a goal of developing new therapies for these challenging diseases.

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

Transcutaneous immunization with a highly active form of XCL1 as a vaccine adjuvant using a hydrophilic gel patch elicits long-term CD8+ T cell responses

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

Precision COVID-19 Vaccine with Companion Diagnostics

&lt;img src=” https://s3.amazonaws.com/production.scholastica/article/12561/medium/prnano_412020_figure_1.jpg?1586516744”&gt; Although disease enhancement by antibodies has been described for corona and other viruses, nobody can predict whether such antibodies induced by the vaccine will not be harmful, especially after reinfection with a different strain. Alternative vaccines could induce memory T-cell responses, in the absence of antibodies, to kill newly infected cells. Preclinical testing of such precision vaccines is performed in silico without animal experiments since epitopes predicted to bind to three HLA class I molecules of a subject activate cytotoxic T-cell response with more than 80% probability. Data science can be utilized to select the immunogenic vaccine peptides from the coronavirus replicase protein and estimate the immune response rate in an HLA-genotyped population. Employing accessible platform technologies, a set of precision vaccines could be co-developed with an HLA-genotype based companion diagnostic to identify the vaccine that most likely induces responses in the subject. The goal of precision vaccination is to convert the deadly COVID-19 into asymptomatic disease and to avoid the potential risk of disease enhancement. READ ARTICLE (https://precisionnanomedicine.com/article/12561)

Hepatocellular Carcinoma Cells Up-regulate PVRL1, Stabilizing PVR and Inhibiting the Cytotoxic T-Cell Response via TIGIT to Mediate Tumor Resistance to PD1 Inhibitors in Mice

Immune checkpoint inhibitors are effective in the treatment of some hepatocellular carcinomas (HCCs), but these tumors do not always respond to inhibitors of programmed cell death 1 (PDCD1, also called PD1). We investigated mechanisms of resistance of liver tumors in mice to infiltrating T cells. Mice were given hydrodynamic tail vein injections of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) and transposon vectors to disrupt Trp53 and overexpress C-Myc (Trp53KO/C-MycOE mice). Pvrl1 and Pvrl3 were knocked down in Hepa1-6 cells by using short hairpin RNAs. Hepa1-6 cells were injected into livers of C57BL/6 mice; some mice were given intraperitoneal injections of antibodies against PD1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or CD8 before the cancer cells were injected. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction; tumors were analyzed by mass cytometry using markers to detect T cells and other lymphocytes. We obtained HCC and nontumorous liver tissues and clinical data from patients who underwent surgery in Hong Kong and analyzed the tissues by immunohistochemistry. Trp53KO/C-MycOE mice developed liver tumors in 3-5 weeks; injections of anti-PD1 did not slow tumor development. Tumors from mice given anti-PD1 had larger numbers of memory CD8+ T cells (CD44+CD62L-KLRG1int) and T cells that expressed PD1, lymphocyte activating 3 (LAG3), and TIGIT compared with mice not given the antibody. HCC tissues from patients had higher levels of PVRL1 messenger RNA and protein than nontumorous tissues. Increased PVRL1 was associated with shorter times of disease-free survival. Knockdown of Pvrl1 in Hepa1-6 cells caused them to form smaller tumors in mice, infiltrated by higher numbers of CD8+ T cells that expressed the inhibitory protein TIGIT; these effects were not observed in mice with depletion of CD8+ T cells. In Hepa1-6 cells, PVRL1 stabilized cell surface PVR, which interacted with TIGIT on CD8+ T cells; knockdown of Pvrl1 reduced cell-surface levels of PVR but not levels of Pvr messenger RNA. In Trp53KO/C-MycOE mice and mice with tumors grown from Hepa1-6 cells, injection of the combination of anti-PD1 and anti-TIGIT significantly reduced tumor growth, increased the ratio of cytotoxic to regulatory T cells in tumors, and prolonged survival. PVRL1, which is up-regulated by HCC cells, stabilizes cell surface PVR, which interacts with TIGIT, an inhibitory molecule on CD8+ effector memory T cells. This suppresses the ant-tumor immune response. Inhibitors of PVRL1/TIGIT, along with anti-PD1 might be developed for treatment of HCC.

A T Cell Receptor Sequencing-Based Assay Identifies Cross-Reactive Recall CD8+ T Cell Clonotypes Against Autologous HIV-1 Epitope Variants.

HIV-1 positive elite controllers or suppressors control viral replication without antiretroviral therapy, likely via CTL-mediated elimination of infected cells, and therefore represent a model of an HIV-1 functional cure. Efforts to cure HIV-1 accordingly rely on the existence or generation of antigen-specific cytotoxic T lymphocytes (CTL) to eradicate infected cells upon reversal of latency. Detecting and quantifying these HIV-1-specific CTL responses will be crucial for developing vaccine and T cell-based immunotherapies. A recently developed assay, called MANAFEST, uses T cell receptor (TCR) Vβ sequencing of peptide-stimulated cultures followed by a bioinformatic pipeline to identify neoantigen-specific T cells in cancer patients. This assay is more sensitive than conventional immune assays and therefore has the possibility to identify HIV-1 antigenic targets that have not been previously explored for vaccine or T cell immunotherapeutic strategies. Here we show that a modified version of the MANAFEST assay, called ViraFEST, can identify memory CD8+ T cell responses against autologous HIV-1 Gag and Nef epitope variants in an elite suppressor. Nine TCR Vβ clonotypes were identified and 6 of these were cross-reactive for autologous variants or known escape variants. Our findings are a proof of principle that the ViraFEST assay can be used to detect and monitor these responses for downstream use in immunotherapeutic treatment approaches.

Streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging

Identification of tumor antigens that induce cytotoxic T lymphocytes (CTLs) is crucial for cancer-vaccine development. Despite their predictive ability, current algorithmic approaches and human leukocyte antigen (HLA)-peptidomic analysis allow limited selectivity. Here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger CTL responses. We used a combined application of this method involving immune-specific signature analysis and HLA-associated peptidomics using samples from six patients with triple-negative breast cancer (TNBC) in order to select immunogenic HLA epitopes for in vitro testing. Additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. The results indicated that this method enabled identification of promising CTL peptides capable of inducing antitumor immunity. This platform combining high-resolution computational analysis, HLA-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic Tcell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immunecells, including Tcells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by Tcells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Abstract B87: NK cells lose their ability to control MHC-I-deficient tumor cells outside circulation

Abstract A common tumor immune-evasion mechanism is downregulation of major histocompatibility complex Class-I (MHC-I) that enables malignant cell escape from cytotoxic T-cell responses. This strategy also contributes to nonresponsiveness of tumors to T-cell targeted immune-checkpoint blockade. MHC-I is an antigen-presenting molecule that also functions as an inhibitory ligand for natural killer (NK) cells. Thus, loss of MHC-I should render cells susceptible to NK cell-mediated cytotoxicity. However, this does not always occur. Previously it was demonstrated that NK cells were able to strongly reject MHC-I-deficient bone marrow cells in the circulation, yet they failed to reject MHC-I-deficient organ grafts, suggesting restricted NK cell cytotoxicity in tissues. We hypothesized that a similar NK cell tolerance to MHC-I-deficient cells occurs in the context of solid cancers. We compared NK cell recruitment and activation using WT and MHC-I-deficient B16F10 melanoma models. We demonstrated that NK cells eradicate circulating MHC-I−/− B16F10 cells, effectively preventing tumor metastasis to the lungs. In contrast, NK cells failed to clear MHC-I−/− B16F10 tumors when implanted subcutaneously, nor was the growth of these tumors affected by NK cell depletion. Interestingly, spontaneous lymph node (LN) metastasis from MHC-I-deficient solid tumors was fully abrogated in an NK cell-dependent manner, indicating functional inhibition of NK cells in peripheral tissues, but not in circulation or draining lymphatics. In conclusion, we demonstrate NK cell tolerance to MHC-I-deficient tumor cells in tissues, but not in circulation, which suggests the presence of non-MHC-I NK cell-inhibitory signals in solid organs. Our research uncovers a fundamental aspect of NK cell biology and will open up the possibility of new NK cell-based therapeutic strategies to control solid tumors that escape cytotoxic CD8+ T-cell responses by downregulating MHC-I. Citation Format: Marta Requesens, Mark Bunting, Maulik Vyas, Shawn Demehri. NK cells lose their ability to control MHC-I-deficient tumor cells outside circulation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B87.

Abstract B63: Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma

Abstract Electrochemotherapy is an established treatment modality in which chemotherapeutics are delivered systemically or locally in combination with local electrical pulses. Pulsing parameters are optimized to induce the reversible formation of pores in the cell membrane, leaving cells intact and healthy. Osmotic gradients during poration result in the rapid uptake of extracellular components, which results in enhanced chemotherapeutic uptake, between 5 to 2,000-fold depending on the hydrophobicity of the drug used. Complete local response rates for dermal lesions exceed 80%. Treatment is suitable for use in proximity to vasculature and offers superior cosmetic results in comparison to surgery when used in areas such as the eyelid or mouth. Recent advances have, however, been able to replace cytotoxic chemotherapeutic agents with calcium with no decrease in efficacy. The nature of cell death induced is believed to be acute, but to date has not been clearly characterized. Using Flou-4 to track the duration of elevated intracellular calcium concentrations, we have shown that elevated calcium levels persist for hours following treatment. Cell death occurs in 60-80% of cells treated in vitro within 8 hours of treatment. Cells surviving this initial insult show no detectable loss in survival or growth potential and appear otherwise unaffected. Necrotic cell death provides a significant source of damage-associated molecular patterns and may aid the generation of anticancer immune responses. Using cocultured macrophages, we have been able to show that treatment can indirectly boost cytotoxic T-cell responses via tumor-associated macrophages. The elucidation of cell death during treatment is critical for the effective optimization of clinical treatment parameters, which has hitherto been determined empirically. Other advances, such as the use of high-frequency electroporation, are transforming the clinical delivery. It is foreseeable that the requirement of surgical theaters may be avoided with the development of a pain-free treatment in the absence of cytotoxic drugs. Note:This abstract was not presented at the conference. Citation Format: Liam Friel Tremble, Cynthia Heffron, Patrick Forde. Calcium electroporation offers an effective local treatment with immunogenic cell death and the potential for systemic remission in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B63.

Immunological memory and tolerance at the maternal-fetal interface: Implications for reproductive management of mares

The development of placentation that coincided with the evolution of mammals presented new challenges to the transmission of life from one generation to the next, particularly with regard to the possibility of maternal immunological recognition and destruction of the developing conceptus. The balance between immunity and tolerance dominates the immunological relationship between mother and fetus during mammalian pregnancy, and the focal point of this relationship lies at the interface between the trophoblast cells that comprise the outermost layer of the placenta and the maternal endometrial tissues. Immune memory and tolerance are two of the cardinal characteristics of the immune system. Immune memory is essential in preventing or lessening the effect of infections to the mother or conceptus, but may also be a threat to the semi-allogeneic tissues of the fetus and placenta. The mother must develop functional immune tolerance to her fetus, but at the same time retain her ability to combat infections while pregnant. To address this imperative, mammals have developed overlapping and independent mechanisms for evading maternal anti-fetal immune responses that could result in pregnancy loss. Studies of the unusual component of equine invasive trophoblast in the epitheliochorial placenta have illuminated aspects of immune memory and tolerance that have relevance to fertility in the horse and other mammalian species.

Distribution of BCG-CWS-Loaded Nanoparticles in the Spleen After Intravenous Injection Affects Cytotoxic T Lymphocyte Activity

Interest has developed in the bacillus Calmette-Guerin (BCG) cell wall skeleton (BCG-CWS) as a noninfectious adjuvant. Although BCG-CWS readily undergoes aggregation, in a previous study, we applied it to cancer immunotherapy via intravenous administration by encapsulating the BCG-CWS into nanoparticles (CWS-NPs). The CWS-NPs were taken up by major histocompatibility complex (MHC) class II+ (MHC-II+) cells and induced antigen-specific cytotoxic T lymphocyte (CTL) activity. However, the nature of the contribution of MHC-II+ cells to the CTL response continues to be unclear. In this study, we investigated the relationship between the distribution of CWS-NPs in the spleen and CTL activity. The main MHC-II+ cells that internalized the CWS-NPs were B cells. Decreasing the level of polyethylene glycol modification increased the uptake of CWS-NPs by B cells, leading to an increased CTL activity. A comparison of CWS-NPs with different uptake efficiencies into dendritic cells and B cells suggested that the DCs with internalized CWS-NPs may contribute to CTL activation compared with B cells. We succeeded in enhancing CTL activity by the CWS-NPs, and the findings reported herein should provide important information regarding target cells for the development of CWS-NP.

Mixed 20-peptide cancer vaccine in combination with docetaxel and dexamethasone for castration-resistant prostate cancer: a randomized phase II trial

A novel cancer vaccine consisting of 20 mixed peptides (KRM-20) was designed to induce cytotoxic T lymphocytes (CTL) against twelve different tumor-associated antigens. The aim of this phase II trial was to examine whether KRM-20 in combination with docetaxel and dexamethasone enhances the antitumor effects in patients with castration-resistant prostate cancer (CRPC). In this double-blind, placebo-controlled, randomized phase II study, we enrolled chemotherapy-naïve patients with CRPC from ten medical centers in Japan. Eligible patients were randomly assigned 1:1 centrally to receive either KRM-20 combined with docetaxel and dexamethasone (n = 25) or placebo with docetaxel and dexamethasone (n = 26). The primary endpoint was the difference in prostate-specific antigen (PSA) decline between each treatment. The rates of > 50% PSA decline in the two arms were similar (56.5% versus 53.8%; P = 0.851). Human leukocyte antigen (HLA)-matched peptide-specific immunoglobulin G (P = 0.018) and CTL (P = 0.007) responses in the KRM-20 arm significantly increased after treatment. The addition of KRM-20 did not increase toxicity. There were no between-group differences in progression-free or overall survival (OS). The addition of KRM-20 was safe, and similar PSA decline and HLA-matched peptide-specific CTL and IgG responses increased in combination with docetaxel and dexamethasone in CRPC patients. Subgroup analysis suggested that this treatment is favorable for CRPC patients with ≥ 26% lymphocytes or PSA levels of < 11.2 ng/ml, but further clinical trials comparing OS are required.