Levels Of HLA-DR Research Articles

Defective Severe Acute Respiratory Syndrome Coronavirus 2 Immune Responses in an Immunocompromised Individual With Prolonged Viral Replication.

We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2–specific antibody responses were absent and SARS-CoV-2–specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.

HHLA2 Expression is Associated with Poor Survival in Patients with Hepatocellular Carcinoma.

BackgroundHuman endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) is a member of the B7 family; however, little is known regarding its expression and clinical relevance in hepatocellular carcinoma (HCC).MethodsTo better characterize HHLA2 expression in HCC, we analyzed its expression by in situ staining and further investigated its correlation with immune infiltration and patient prognosis.ResultsHHLA2 was primarily expressed in the peri-tumor region of HCC tissues and co-localized with CD68+ monocytes/macrophages. In vitro analysis and multi-immunofluorescence staining showed up-regulated HHLA2 expression in tumor-activated monocytes/macrophages, and HHLA2+ monocytes/macrophages expressed high levels of HLA-DR in HCC tissue. A correlation analysis showed that samples displaying high HHLA2 expression in the peri-tumor region had significant tumor infiltration of CD204+ and CD11b+ cells, and low expression of genes associated with an anti-tumor immune response. The high level of peri-tumoral HHLA2 expression was associated with a poor patient overall survival (OS; P = 0.008). A multivariate analysis revealed that HHLA2 expression in the peri-tumor region was an independent prognostic factor for OS (hazard ratio = 1.872, p = 0.003). Moreover, the expression of HHLA2 was negatively correlated with PD-L1, and patients exhibiting HHLA2 and programmed cell death-ligand 1(PD-L1) co-expression had the shortest survival time.ConclusionHHLA2 expression represented an immunosuppressive microenvironment in HCC, and may serve as a potential target for immunotherapy.

Expression of HLA-DR in Cytotoxic T Lymphocytes: A Validated Predictive Biomarker and a Potential Therapeutic Strategy in Breast Cancer.

Simple SummaryMore than 50% of breast cancer (BC) patients selected for neoadjuvant chemotherapy (NACT) are subjected to at least a 6-month regimen of this treatment without a clear benefit, probably delaying more effective therapeutic strategies and being exposed to potential treatment-associated toxicity. Thus, it is urgent to implement reliable predictive biomarkers, as well as novel treatments for NACT non-responder patients. This study validates that the HLA-DR level in cytotoxic T lymphocytes (CTLs) is an independent and robust predictive factor of BC patients’ response to NACT, as previously proposed. Hence, a predictive probability model of response was developed as a new tool to improve treatment decisions. HLA-DR level in CTLs also have a general prognostic value, which might be relevant for long-term BC management. In addition, our results suggest that increasing the expression of HLA-DR in CTLs of non-responders could be a promising therapeutic strategy to ameliorate BC response to NACT.Neoadjuvant chemotherapy (NACT) is common in breast cancer (BC) treatment, though more than half of the patients lack an effective response. Therefore, new predictive biomarkers and alternative therapies are crucial. Previously, we proposed HLA-DR-expressing cytotoxic T lymphocytes (CTLs) as a potential biomarker of the response to NACT. To validate this observation and further investigate these cells, 202 BC patients were enrolled. Flow cytometry analyses were performed in 61 biopsies and 41 blood samples pre-NACT and 100 non-NACT tumor samples. All the patients were followed up for 34 months. Blood-isolated immune cells were cultured with BC cell lines in a 3D system. We confirmed that HLA-DR level in CTLs is a highly sensitive, specific, and independent biomarker to predict response to NACT and developed a predictive probability model. This biomarker was also associated with progression-free survival, regardless of the treatment. The clinical observations are substantiated by the anti-tumor properties of HLA-DR-expressing CTLs. Intriguingly, HLA-DR level in CTLs can be modulated ex vivo, boosting their capacity to kill tumor cells synergistically with doxorubicin. Thus, HLA-DR expression in CTLs is a validated tool to select patients that will actually benefit from NACT, and its stimulation might be a novel therapeutic approach for BC.

Multidimensional analysis of immune responses identified biomarkers of recent Mycobacterium tuberculosis infection.

The risk of tuberculosis (TB) disease is higher in individuals with recent Mycobacterium tuberculosis (M.tb) infection compared to individuals with more remote, established infection. We aimed to define blood-based biomarkers to distinguish between recent and remote infection, which would allow targeting of recently infected individuals for preventive TB treatment. We hypothesized that integration of multiple immune measurements would outperform the diagnostic performance of a single biomarker. Analysis was performed on different components of the immune system, including adaptive and innate responses to mycobacteria, measured on recently and remotely M.tb infected adolescents. The datasets were standardized using variance stabilizing scaling and missing values were imputed using a multiple factor analysis-based approach. For data integration, we compared the performance of a Multiple Tuning Parameter Elastic Net (MTP-EN) to a standard EN model, which was built to the individual adaptive and innate datasets. Biomarkers with non-zero coefficients from the optimal single data EN models were then isolated to build logistic regression models. A decision tree and random forest model were used for statistical confirmation. We found no difference in the predictive performances of the optimal MTP-EN model and the EN model [average area under the receiver operating curve (AUROC) = 0.93]. EN models built to the integrated dataset and the adaptive dataset yielded identically high AUROC values (average AUROC = 0.91), while the innate data EN model performed poorly (average AUROC = 0.62). Results also indicated that integration of adaptive and innate biomarkers did not outperform the adaptive biomarkers alone (Likelihood Ratio Test χ2 = 6.09, p = 0.808). From a total of 193 variables, the level of HLA-DR on ESAT6/CFP10-specific Th1 cytokine-expressing CD4 cells was the strongest biomarker for recent M.tb infection. The discriminatory ability of this variable was confirmed in both tree-based models. A single biomarker measuring M.tb-specific T cell activation yielded excellent diagnostic potential to distinguish between recent and remote M.tb infection.

Effective and Ineffective Samples of Mesenchymal Multipotent Stromal Cells Used for Acute Graft-Versus-Host Disease Prophylaxis after Allogeneic Bone Marrow Transplantation Differ in the Expression Level of HLA-DR When Co-Cultured with Activated Lymphocytes

IntroductionMultipotent mesenchymal stromal cells (MSCs) are widely used for the prevention and treatment of acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT). In some cases prophylaxis of aGVHD failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or differences in MSC samples. In order to discriminate effective and ineffective for aGVHD prophylaxis MSCs the growth and differentiation characteristics, relative expression levels of different genes and changes in the expression of surface MSC molecules during co-cultivation with activated lymphocytes were investigated in all MSC samples.MethodsMSCs from the bone marrow of corresponding hematopoietic stem cells donor were injected into 59 patients after allo-BMT at the moment of blood count recovery. MSCs from the bone marrow of those donors were analyzed by cultivation in standard conditions (aMEM, 10% fetal calf serum) for 3 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR in all MSC samples. MSCs were seeded 105 cells per T25 flask, and then 106 allogeneic lymphocytes from single donor were added to all MSCs cultures. For lymphocytes activation 5 mg/ml phytohemagglutinin (PHA) was added to half of these cultures. Lymphocytes were removed from MSCs on 1st and 4th days of co-cultivation. Than trypsinized MSCs were collected from the bottom of the flask and expression of HLA-DR, HLA-ABC, CD73, PD-L1, CD54, CD80, CD86, CD105, CD146 and FAS-L on their surface was measured by flow cytometry. p<0.05 was considered statistically significant, all data is present as medium ± SEM.ResultsThe injection of MSCs reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (p=0.047). Eight of 51 MSC samples had been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) found no significant differences between the MSCs, effective and ineffective for preventing aGVHD. REL analysis of 30 genes involved in proliferation, differentiation and immunomodulation in MSCs reviled only 4 genes differs significantly between effective and ineffective MSC samples (table). During 4 days of cultivation the MFI of HLA-DR did not change on MSCs. When MSCs co-cultivated with non-activated lymphocytes for 4 days MFI of HLA-DR increased only in 1.3 fold (p=0.03). Co-cultivation of MSCs with activated lymphocytes led to 7 fold increases in HLA-DR expression between 1st and 4th days (p≤0,001). On 4th day of co-cultivation MSCs with activated lymphocytes the MFI of HLA-DR was 3 fold lower in ineffective MSC samples than in effective (table). These data indicate that the effectiveness of MSCs used depends on the degree of their immunogenicity and, accordingly, on the immunomodulating potential. Among studied molecules only Fas L was significantly lower in effective MSC samples than in ineffective (table). It is possible that such MSC samples survived in the organism better than ineffective and their immunomodulating function lasts longer in the organism.ConclusionsIn summary, this study showed that MSC injections resulted in a significant 2-fold decrease in aGvHD development compared with the standard prophylaxis treatment. When compared with ineffective MSC, effective MSC samples are characterized by higher total cell production and REL of CFH, FGFR1, PDGFRa, and ICAM1. ExpressionofHLA-DR increased significantly in MSCs co-cultivated with lymphocytes, so these cells became immunogenic in organism. It is evident that the level of HLA-DR expression on MSCs important for MSCs efficiency in aGVHD prophylaxis. It is possible to predict efficiency of MSCs injection by simple test of co-cultivation of these cells with activated lymphocytes and measuring the expression level of HLA-DR on them.The materials are supported by grant from the Russian Science Foundation, Project № 16-15-00102 [Display omitted] DisclosuresShipounova:Russian Foundation for Basic Research, Project № 15-04-02514: Research Funding.

Dynamic changes in peripheral blood lymphocyte subset counts and functions in patients with diffuse large B cell lymphoma during chemotherapy

BackgroundThis study aimed to analyze the lymphocyte subsets, their activities and their dynamic changes during immunochemotherapy in patients newly diagnosed with diffuse large B cell lymphoma (DLBCL).MethodsPatients with DLBCL (n = 33) were included in the present study. Their peripheral lymphocyte subsets, phenotypes and functions were detected using flow cytometry. The dynamic results of lymphocyte activities were available for 18 patients.ResultsCompared with healthy controls (HCs), the counts of CD3+, CD4+, and CD8+ T cells as well as those NK cells decreased in patients newly diagnosed with DLBCL, mainly attributed to patients with high risk of prognosis assessed by International Prognostic Index (IPI) score. Lymphocyte counts didn’t present significant difference between high risk (IPI scores 3–5) and low risk patients (IPI scores 0–2), but CD4+ T cells and CD8+ T cells expressed higher levels of CD28 and HLA-DR, respectively, in patients with IPI score ranging from 3 to 5. Patients at high risk harbored higher percentage of regulatory T cells (Tregs), and their CD4+ and CD8+ T cells produced lower levels of IFN-γ, reflecting an impaired cellular immune response. The dynamic changes of lymphocyte numbers and functions during treatment were further investigated. Total counts of CD3+, CD4+, CD8+ T and NK cells progressively decreased because of the cytotoxicity of chemotherapy and then gradually recovered after six cycles treatment (rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisone, R-CHOP). The functions of CD4+ and CD8+ T cells recovered by the end of two cycles R-CHOP treatment, although NK cell function was not significantly affected throughout treatment. These results suggest that the counts and functions of lymphocytes are significantly decreased in patients with DLBCL, particularly those of CD4+ and CD8+ T cells.ConclusionsThe absolute counts and functions of CD4+, CD8+ T cells, which were significantly lower in patients with DLBCL, gradually recovered after effective treatment. Therefore, combined detection of T cell counts and functions are critically important for administering effective personalized immunotherapy as well as for identifying new prognostic markers or DLBCL.

Rapid progression is associated with lymphoid follicle dysfunction in SIV-infected infant rhesus macaques.

HIV-infected infants are at an increased risk of progressing rapidly to AIDS in the first weeks of life. Here, we evaluated immunological and virological parameters in 25 SIV-infected infant rhesus macaques to understand the factors influencing a rapid disease outcome. Infant macaques were infected with SIVmac251 and monitored for 10 to 17 weeks post-infection. SIV-infected infants were divided into either typical (TypP) or rapid (RP) progressor groups based on levels of plasma anti-SIV antibody and viral load, with RP infants having low SIV-specific antibodies and high viral loads. Following SIV infection, 11 out of 25 infant macaques exhibited an RP phenotype. Interestingly, TypP had lower levels of total CD4 T cells, similar reductions in CD4/CD8 ratios and elevated activation of CD8 T cells, as measured by the levels of HLA-DR, compared to RP. Differences between the two groups were identified in other immune cell populations, including a failure to expand activated memory (CD21-CD27+) B cells in peripheral blood in RP infant macaques, as well as reduced levels of germinal center (GC) B cells and T follicular helper (Tfh) cells in spleens (4- and 10-weeks post-SIV). Reduced B cell proliferation in splenic germinal GCs was associated with increased SIV+ cell density and follicular type 1 interferon (IFN)-induced immune activation. Further analyses determined that at 2-weeks post SIV infection TypP infants exhibited elevated levels of the GC-inducing chemokine CXCL13 in plasma, as well as significantly lower levels of viral envelope diversity compared to RP infants. Our findings provide evidence that early viral and immunologic events following SIV infection contributes to impairment of B cells, Tfh cells and germinal center formation, ultimately impeding the development of SIV-specific antibody responses in rapidly progressing infant macaques.

Letermovir Administration to Prevent Cytomegalovirus Reactivation Is the Potential Risk of Chronic Graft-Versus-Host Disease in Patients Who Received Haploidentical Stem-Cell Transplantation With Post-Transplant Cyclophosphamide.

The prevention of chronic graft-versus-host disease (cGVHD) is important for recipients of hematopoietic stem-cell transplantation (HSCT). As one of the etiologies, the relationship between early T-cell recovery and subsequent cGVHD development has been the focus of attention. Recently, letermovir (LTV) was approved for preventing cytomegalovirus (CMV) reactivation in the early transplantation phase. Although CMV affects the immune reconstitution after HSCT, the impacts of LTV to prevent CMV reactivation on early T-cell recovery and cGVHD have not been fully investigated. We aimed to identify early T-cell recovery under LTV at day 30 in 15 and 33 recipients from matched related donors (MRDs) and haploidentical donors with post-transplant cyclophosphamide (PTCy-haplo), respectively. Early increases in the levels of total lymphocytes and HLA-DR+ activated T-cells at day 30 were observed under CMV prophylaxis by LTV only in PTCy-haplo recipients and not in MRD recipients. Moreover, PTCy-haplo recipients with LTV showed a significantly higher incidence of cGVHD, but not acute GVHD. Our observations suggest that an early increase in the levels of HLA-DR+ activated T-cells may be implicated in the development of cGVHD in patients treated with PTCy who received LTV. Further studies are warranted to validate our results and elucidate the detailed mechanisms of our new insights.

In-vitro Immunomodulatory activity of Azadirachta indica A.Juss. Ethanol: water mixture against HIV associated chronic CD4+ T-cell activation/ exhaustion

BackgroundIn Sub-Saharan Africa, herbal therapy continues to be utilized for HIV-1 disease management. However, the therapeutic benefits of these substances remain ambiguous. To date, little is known about the effects of these plant extracts on chronic CD4 + T-cell activation and exhaustion which is partly driven by HIV-1 associated microbial translocation.MethodsEffects of Azadirachta indica, Momordica foetida and Moringa oleifera ethanol: water mixtures on cell viability were evaluated using the Guava PCA system. Then, an in-vitro cell culture model was developed to mimic CD4+ T cell exposures to antigens following HIV-1 microbial translocation. In this, peripheral blood mononuclear cells (PBMCs) isolated from HIV negative (n = 13), viral load < 1000 copies per mL (n = 10) and viral load > 1000 copies per mL (n = 6) study participants from rural Uganda were treated with Staphylococcus enterotoxin B (SEB). Then, the candidate plant extract (A. indica) was added to test the potential to inhibit corresponding CD4+ T cell activation. Following BD Facs Canto II event acquisition, variations in %CD38, %CD69, Human Leukocyte Antigen -DR (HLA-DR), Programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), interferon gamma (IFN γ) and interleukin 2 (IL-2) CD4 + T cell expression were evaluated.ResultsFollowing exposure to SEB, only A. indica demonstrated a concentration-dependent ability to downregulate the levels of CD4 + T cell activation. At the final concentration of 0.500 μg/mL of A. indica, a significant downregulation of CD4 + CD38 + HLA-DR+ expression was observed in HIV negative (p < 0.0001) and both HIV infected groups (P = 0.0313). This plant extract also significantly lowered SEB induced % CD4+ T cell HLADR, PD-1 and Tim-3 levels. PD-1 and CD69 markers were only significantly downmodulated in only the HIV negative ((p = 0.0001 and p = 0.0078 respectively) and viral load< 1000 copies per ml (p = 0.0078) groups.ConclusionA. indica exhibited the in-vitro immunomodulatory potential to inhibit the continuum of SEB induced CD4+ T-cell activation/ exhaustion without impacting general T-cell specific functions such as cytokine secretion. Additional studies are needed to confirm A. indica as a source of natural products for targeting persistent immune activation and inflammation during ART.

ONCOLYSIS MEDIATED BY INFB AND ARF GENE TRANSFER ACTIVATES KEY COMPONENTS OF THE IMMUNE SYSTEM

<h3>Background</h3> The importance of the immune system for controlling neoplasms was highlighted with the success of checkpoint blockade therapy. New cancer therapeutics seek to break the immune tolerance inherent in tumor progression. To this end, some virus-mediate gene therapy approaches have been shown to induce oncolysis accompanied by emission of immunogenic cell death (ICD) factors, thus attracting and activating cells of the immune system. <h3>Objective</h3> To use combined adenovirus-mediated gene transfer of p14ARF and human interferon-β (IFNβ) to human melanoma cells to induce oncolysis, ICD and subsequent activation of immune cells. <h3>Methods</h3> Cell death was measured by PI and/or AnnexinV staining. Detection of calreticulin was performed by cytometry, while ATP and IFNβ were detected by ELISA. DC and lymphocytes from healthy donors were cultivated ex vivo. <h3>Results</h3> IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. However, detection of critical markers of ICD) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. In situ gene therapy for s.c. SK-MEL-147 tumors in Nod-Scid mice controlled tumor growth and increased animal survival in response to IFNβ alone or in combination with p14ARF. However, the immunocompromised animal model cannot demonstrate the expected immune activation. Thus, we co-cultured SK-MEL-147 transduced with our combination of adenoviruses with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors. These Mo-DCs showed increased levels of activation markers HLA-DR, CD80, and CD86. Next, we used these activated Mo-DCs to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. T cells primed in this way were co-cultured with fresh non-transduced SK-MEL-147, resulting in tumor cell killing. Currently we are expanding these studies to include three-dimensional cultures of melanoma cell lines and also the use of human melanoma samples in patient derived organoid (PDO) models. We expect to reveal the influence of oncolysis on tumor infiltrating cells, including DCs, macrophages, NK and T cells. <h3>Conclusion</h3> Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Monocyte subset redistribution from blood to kidneys in patients with Puumala virus caused hemorrhagic fever with renal syndrome.

Innate immune cells like monocytes patrol the vasculature and mucosal surfaces, recognize pathogens, rapidly redistribute to affected tissues and cause inflammation by secretion of cytokines. We previously showed that monocytes are reduced in blood but accumulate in the airways of patients with Puumala virus (PUUV) caused hemorrhagic fever with renal syndrome (HFRS). However, the dynamics of monocyte infiltration to the kidneys during HFRS, and its impact on disease severity are currently unknown. Here, we examined longitudinal peripheral blood samples and renal biopsies from HFRS patients and performed in vitro experiments to investigate the fate of monocytes during HFRS. During the early stages of HFRS, circulating CD14-CD16+ nonclassical monocytes (NCMs) that patrol the vasculature were reduced in most patients. Instead, CD14+CD16- classical (CMs) and CD14+CD16+ intermediate monocytes (IMs) were increased in blood, in particular in HFRS patients with more severe disease. Blood monocytes from patients with acute HFRS expressed higher levels of HLA-DR, the endothelial adhesion marker CD62L and the chemokine receptors CCR7 and CCR2, as compared to convalescence, suggesting monocyte activation and migration to peripheral tissues during acute HFRS. Supporting this hypothesis, increased numbers of HLA-DR+, CD14+, CD16+ and CD68+ cells were observed in the renal tissues of acute HFRS patients compared to controls. In vitro, blood CD16+ monocytes upregulated CD62L after direct exposure to PUUV whereas CD16- monocytes upregulated CCR7 after contact with PUUV-infected endothelial cells, suggesting differential mechanisms of activation and response between monocyte subsets. Together, our findings suggest that NCMs are reduced in blood, potentially via CD62L-mediated attachment to endothelial cells and monocytes are recruited to the kidneys during HFRS. Monocyte mobilization, activation and functional impairment together may influence the severity of disease in acute PUUV-HFRS.

Expression levels of HLA-DR in acute myeloid leukemia: implications for antigenicity and clinical outcome

Low human leukocyte antigen (HLA)-DR expression might compromise CD4+ T-cell-mediated anti-tumor immunity. Its immunological and clinical significance however remain undefined in non-promyelocytic acute myeloid leukemia (AML). Taking advantage of mass spectrometry-based immunopeptidome analysis of primary AML samples (n = 31), we studied the implications of low HLA-DR expression for antigen presentation and analyzed its association with disease characteristics and survival within a cohort of 399 AML patients. Remarkably, overall HLA-DR/DQ immunopeptidome diversity was preserved in AML with low HLA-DR expression (HLA-DRlow AML) and was associated with a shift in HLA-DR/DQ allotype abundances (HLA-DQ to HLA-DR/DQ ligand ratio 0.36 vs 0.19 in HLA-DRlow and HLA-DRhigh patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DRlow and HLA-DRhigh patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DRlow AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DRlow AML patients.

P14.20 Multi-Spectral Imaging of Lung Adenocarcinoma Reveals Importance of Cancer Specific HLA-DR on the TME and Clinical Outcome

Within the tumor microenvironment (TME), the interactions between cancer cells and immune cells are of increasing importance as targeting key immune checkpoints have redefined clinical care for patients with lung cancer. While immune checkpoint inhibitors have improved outcomes for patients with NSCLC, only a subset of patients respond. Tremendous efforts have been made to define factors that mediate tumor progression and response to immunotherapy. Regulation of antigen-presentation has been shown to play a critical role in this response. MHCII (HLA-DR) is normally expressed on antigen presenting cells and leads to activation of CD4+ T cells. Notably, HLA-DR expression is observed on cancer cells and appears to shape local anti-tumor immunity through potential direct presentation of tumor neoantigens to CD4+ helper T cells. While several studies have demonstrated that HLA-DR on cancer cells leads to more T cell infiltration, its effect on the TME and outcomes for patients with NSCLC have not been systemically examined. Advances in multispectral immunohistochemistry techniques allow for quantitative and spatial identification of multiple immune cells. Imaging with multiple markers can reveal the diversity of the local immune infiltrate, cell-cell interactions, and correlations of spatial distribution and location of immune cells in NSCLC with clinical outcome of patients with cancer cells. Slides from 153 patients with resected lung adenocarcinoma were stained using Opal multiplex with a panel of markers: DAPI, HLA-DR, CK, CD3, CD14, CD19 and CD8. Multispectral imaging was performed using wide field microscopy on the Vectra3.0. For each patient, 3-5 regions of interest (ROIs) were chosen and spectral unmixing was utilized for the detection and separation of seven different fluorescent signals in each NSCLC tissue samples. Cells were segmented based on nuclear and membrane markers and classified in phenotypes using inForm machine learning algorithms. The immunophenotypes were quantified and nearest neighbor analysis was performed. The presence of HLA-DR on cancer cells and immune cell infiltration were correlated with clinical outcome. Co-expression of HLA-DR and CK+ defined cancer cells that express MHCII. HLA-DR high and HLA-DR low patients were stratified using a cutoff of >5% of cancer cells expressing HLA-DR. Patients with HLA-DR high tumors had significantly increased immune cell infiltration of CD4+, CD8+, and CD14+ cells. Nearest neighbor analysis demonstrated CK+ tumor cells in HLA-DR high patients were in closer proximity to CD14+, CD19+, CD4+, and CD8+ T cells. In HLA-DR high patients, more CK+ cancer cells were directly interfaced with CD4+ and CD8+ T cells. Patients with high levels of cancer cell HLA-DR experienced improved overall survival at 5 years (p=0.046). HLA-DR high tumors are significantly associated with increased T cell and myeloid cell infiltration, resulting in closer proximity of all immune cells and more cell-cell contact. Imaging analysis of HLA-DR high tumors demonstrated more direct contact with CD4+ and CD8+ T cells compared to HLA-DR low tumors. Patients with HLA-DR high tumors experienced an improved overall survival compared to patients with HLA-DR low tumors. These data suggest cancer cell HLA-DR may represent a new biomarker for TME inflammation and patient outcomes in NSCLC.

10-hydroxy-2-decenoic acid a bio-immunomodulator in tissue engineering; generates tolerogenic dendritic cells by blocking the toll-like receptor4.

Dendritic cells (DCs), in response to the biomaterials, utilize toll-like receptors (TLRs) to become mature or tolerogenic through TLRs-dependent signaling pathways, especially TLR4. Regarding the physicochemical properties of biomaterials, some of such signaling pathways are activated. Unsaturated fatty acids have been explored as an antagonist for TLRs and lead to the tolerogenic phenotype of DCs. Here we showed that, although cultured DCs on both chitosan and Alginate-polyethyleneimine (Alg-PEI) films became fully mature, 10-hydroxy-2-decanoic acid (10-HDA), an unsaturated fatty acid found in royal jelly, led to the tolerogenic immunophenotype of DCs on both films. The cultured cells on the films possessed iDCs-like morphology in the presence of 10-HDA. Moreover, 10-HDA expressed lower levels of CD80, CD83, CD86, and HLA-DR, a higher level of IL-10, and lower level of IL-12 in the cultured DCs on both films. Furthermore, HEK293T cells expressing only TLR4 (HEK-TLR4 cells) were co-cultured with LPS, a specific agonist for TLR4, and 10-HDA. The 10-HDA significantly reduced the expression of tumor necrosis factor-a (TNF-α) in the HEK-TLR4 cells compared to treated only with LPS. These findings indicate that the 10-HDA acts as an antagonist of TLR4; therefore, potentially can be used in autoimmune diseases and preventing the rejection of biomaterials implantation and allograft transplantation.

Human leukocyte antigen class II quantification by targeted mass spectrometry in dendritic-like cell lines and monocyte-derived dendritic cells

The major histocompatibility complex II (HLA-II) facilitates the presentation of antigen-derived peptides to CD4+ T-cells. Antigen presentation is not only affected by peptide processing and intracellular trafficking, but also by mechanisms that govern HLA-II abundance such as gene expression, biosynthesis and degradation. Herein we describe a mass spectrometry (MS) based HLA-II-protein quantification method, applied to dendritic-like cells (KG-1 and MUTZ-3) and human monocyte-derived dendritic cells (DCs). This method monitors the proteotypic peptides VEHWGLDKPLLK, VEHWGLDQPLLK and VEHWGLDEPLLK, mapping to the α-chains HLA-DQA1, -DPA1 and -DRA1/DQA2, respectively. Total HLA-II was detected at 176 and 248 fmol per million unstimulated KG-1 and MUTZ-3 cells, respectively. In contrast, TNF- and LPS-induced MUTZ-3 cells showed a 50- and 200-fold increase, respectively, of total α-chain as measured by MS. HLA-II protein levels in unstimulated DCs varied significantly between donors ranging from ~ 4 to ~ 50 pmol per million DCs. Cell surface HLA-DR levels detected by flow cytometry increased 2- to 3-fold after DC activation with lipopolysaccharide (LPS), in contrast to a decrease or no change in total HLA α-chain as determined by MS. HLA-DRA1 was detected as the predominant variant, representing > 90% of total α-chain, followed by DPA1 and DQA1 at 3–7% and ≤ 1%, respectively.

BIOM-09. MULTIPLEX ANALYSIS OF CSF EXTRACELLULAR VESICLES OF INTRASPINAL TUMORS

Abstract BACKGROUND: Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. We previously demonstrated that extracellular vesicles (EV) from central nervous system tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. Their implication as biomarkers in tumor disease is under current investigation. It is unclear, however, to what extent cerebrospinal fluid (CSF) EVs from intraspinal tumors are utilizable for diagnostical purposes and how their marker profiles overlap with EVs derived from non tumorous EVs. We analyzed CSF EVs of intraspinal tumors to define CSF EV profiles that allow tumor subtype classification. METHODS: EVs were isolated from CSF of patients suffering from intraspinal meningioma (n=5), ependymoma (n=7) and neurinoma (n=5). Patients suffering from normal pressure hydrocephalus were used as controls (n=5). EVs were analyzed by multiplex bead based assay, immunoblotting, electron microscopy and NTA. RESULTS: CSF EVs were 97.21 ± 3.37nm (intraspinal tumor patients) and 101.6 ± 3.68nm (controls) in sizes and showed vesicular structures by electron microscopy. Particle number were not significantly different between both groups (p = 0.103). Using our 37 protein mutliplex EV profiling kit we found 29 proteins to be expressed in a sufficient manner on CSF EVs. CSF EVs of intraspinal meningioma showed elevated CD62P, HLA-DR, CD40, CD42a and CD45 expression levels, while ependymoma showed decreased levels of CD9, CD63, CD81, whereas neurinomas had elevated levels of SSEA-3 and CD25. CONCLUSION: This is the first comprehensive analysis of CSF EV of intraspinal tumor patients. CSF EV display distinct subpopulations that may allow tumor classification and long-term surveillance. However as tumor-specific EVs may be rare, there is still the need to identify markers that can enrich tumor-specific EVs for molecular profiling.

Dasatinib Promotes the Maturation of Healthy Donors and Chronic Myelogenous Leukemia Patients Derived Dendritic Cells

Dasatinib has been discovered to obtain immunomodulatory effects in addition to the targeting effect towards BCR-ABL in chronic myelogenous leukemia (CML) patients. But the effect of dasatinib on dendritic cells (DCs) is still not fully understood. Objective To investigate the effect of dasatinib on the maturation of monocyte derived dendritic cells (moDCs) from healthy donors (HDs) and CML patients. Method Peripheral blood mononuclear cells (PBMCs) were isolated from HDs (n=14) and CML patients (n=14) who had got remission of MR4.5 with imatinib treatment. The generation of moDCs was completed after 7 days of incubation in DC I medium, and another 3 days of incubation in DC II medium with or without 25nM dasatinib. On the 10th day, the moDCs were harvested and analyzed for the expression of surface markers CD83, CD80, CD86, CD40 and HLA-DR to reflect the maturation of the moDCs by flow cytometry. Results When analyzing the moDCs from all the 14 HDs together, dasatinib significantly enhanced the expression levels of the surface marker CD83 (P=0.039), CD40 (P=0.002) and HLA-DR (P=0.000) on moDCs compared with the control group, but there was no statistically significant difference in CD80 (P=0.073) and CD86 (P=0.897). Differently, when analyzing the moDCs derived from all the 14 patients together, there was no statistically significant difference in the expression levels of CD80 (P=0.086), CD86 (P=0.166), CD83 (P=0.674), CD40 (P=0.574) and HLA-DR (P=0.561) on moDCs between the dasatinib group and the control group. However, moDCs derived from 3 of the 14 patients show the enhanced expression of all the five surface makers with dasatinib administration compared to the control group. Besides, the moDCs derived from 6 of the 14 patients show the enhanced expression of at least one of the five surface markers. Notably, compared with moDCs derived from HD group (n=14), moDCs from patient group (n=14) express lower levels of CD80 (P=0.340), CD86 (P=0.007), CD40 (P=0.010), CD83 (P=0.019) and HLA-DR (P=0.036). Conclusion Dasatinib at the concentration of 25nM can efficiently promote the maturation of HD-derived moDCs in vitro, whereas this effect only occurs in part of the CML patients. And the moDCs from patients generally show much lower levels of the maturation associated surface makers compared with moDCs from HDs. Dasatinib shows potential as a DC adjuvant to be applied in DC-based therapeutic strategies, such as DC vaccine and DC cell-therapy. Disclosures No relevant conflicts of interest to declare.

Phenotypic and Proliferative Analysis of T Cell and Monocyte Populations in Patients with Intermediate/High/Very High-Risk MDS Compared to Age-Matched Healthy Donors

Background: Myelodysplastic syndromes (MDS) have been associated with alterations in the bone marrow microenvironment and include abnormal innate and adaptive immune responses. The extent to which these changes contribute to the pathogenesis of MDS is unclear. The heterogeneous nature of MDS makes it difficult to distinguish changes in intrinsic cell function for microenvironmental alterations in the mutated myeloid compartment. T cells, which are in the lymphoid compartment and less frequently contain MDS mutations, can potentially be used to evaluate immune dysfunction in the presence of malignant myeloid cells. Here we examine the immunophenotypes and proliferative function of T lymphocyte and monocyte populations from higher-risk MDS patients. Methods: Peripheral blood mononuclear cells (PBMC) from patients with IPSS-R Intermediate/High/Very High-risk MDS (herein referred to collectively as higher-risk MDS) and age-matched healthy donors (n = 15 and n = 12, respectively) were polyclonally stimulated with an antibody against CD3 (OKT3) in the presence of IL-2 (50 U/mL). Phenotypic analysis was performed at baseline and 46 hours after the addition of OKT3 and IL-2. T cell proliferation was measured by staining PBMC with proliferation dye (CellTrace Violet) and measuring fold proliferation after eight days in culture. Proliferation statistical comparisons were made by unpaired t-test. Phenotypic parameters were measured by flow cytometry and statistical analysis performed with the Kruskal-Wallis one-way analysis. Results: As previously reported, higher-risk MDS patient T cells had decreased capacity for proliferation when compared to healthy donor counterparts (Figure 1A p = 0.0096). This difference in proliferative capacity was not associated with either the level of PD-1 on the surface of T cells (r = 0.2956) or the level of PD-L1 on monocytes (r = 0.3859) in stimulated PBMCs. Patients with higher-risk MDS tended to have lower PD-L1 surface expression on monocytes compared to healthy donor counterparts, albeit not significantly (Figure 1C p = 0.1). However, PD-1 expression on T cells was comparable between groups both before and after stimulation (Figure 1B CD4 cells: p = 0.58; CD8 cells p = 0.58). This suggests that T cell proliferation from patients with higher-risk MDS might not be inhibited through the PD-1/PD-L1 axis. Of note, higher-risk MDS patient monocytes expressed less CD80 and HLA-DR both pre- and post-stimulation when compared to healthy donors (Figure 1C CD80: p = 0.0001 and 0.037; DR: p = 0.012 and 0.019, respectively), implying a potential dysfunction in the monocytic stimulatory and antigen presentation capacity. PD-L1 surface expression is low at baseline (an average of &amp;lt;5% for both donors and higher-risk MDS); upon stimulation, there is a high correlation between HLA-DR and PD-L1 in healthy donors (p = 0.0003, r = 0.859). Additionally, there is less upregulation of PD-L1 on the surface of monocytic cells in the higher-risk MDS group and subsequently less correlation between PD-L1/HLA-DR (p = 0.03) in stimulated PBMC samples. Taken together, these results show a decreased baseline expression and/or ability to upregulate monocyte activation markers after stimulation. We hypothesize that these changes might contribute to the observation that higher-risk MDS patient T cells are less capable of proliferation. Conclusion: There was no significant difference in PD-1 and PD-L1 expression between higher-risk MDS and healthy donor patients, despite reduced T cell proliferation among patients with MDS. Additionally, decreased levels of CD80 and HLA-DR on intermediate/high-risk MDS patient monocytes suggests a suppressed ability to upregulate activation markers, possibly leading to decreased T cell proliferation. Targeting the PD-1/PD-L1 axis alone may not be therapeutic in patients with higher-risk MDS, and we hypothesize that the immune evasion observed in higher-risk MDS patients may not be due exclusively to increases in inhibitory interactions like PD-L1 and PD-1, but may result from a decrease in stimulatory receptors, such as CD80, and antigen presentation molecules, such as HLA-DR. Disclosures Fields: PersImmune, Inc.: Ended employment in the past 24 months. Ferrari:PersImmune, Inc.: Ended employment in the past 24 months. Tarke:PersImmune, Inc.: Ended employment in the past 24 months. Luger:Arcturus Therapeutics: Current Employment. Lane:PersImmune, Inc.: Current Employment. Vitiello:PersImmune, Inc.: Current equity holder in private company, Ended employment in the past 24 months. Bejar:Aptose Biosciences, Inc: Current Employment, Current equity holder in publicly-traded company.

Combined p14ARF and Interferon-β Gene Transfer to the Human Melanoma Cell Line SK-MEL-147 Promotes Oncolysis and Immune Activation.

Immune evasion is an important cancer hallmark and the understanding of its mechanisms has generated successful therapeutic approaches. Induction of immunogenic cell death (ICD) is expected to attract immune cell populations that promote innate and adaptive immune responses. Here, we present a critical advance for our adenovirus-mediated gene therapy approach, where the combined p14ARF and human interferon-β (IFNβ) gene transfer to human melanoma cells led to oncolysis, ICD and subsequent activation of immune cells. Our results indicate that IFNβ alone or in combination with p14ARF was able to induce massive cell death in the human melanoma cell line SK-MEL-147, though caspase 3/7 activation was not essential. In situ gene therapy of s.c. SK-MEL-147 tumors in Nod-Scid mice revealed inhibition of tumor growth and increased survival in response to IFNβ alone or in combination with p14ARF. Emission of critical markers of ICD (exposition of calreticulin, secretion of ATP and IFNβ) was stronger when cells were treated with combined p14ARF and IFNβ gene transfer. Co-culture of previously transduced SK-MEL-147 cells with monocyte-derived dendritic cells (Mo-DCs) derived from healthy donors resulted in increased levels of activation markers HLA-DR, CD80, and CD86. Activated Mo-DCs were able to prime autologous and allogeneic T cells, resulting in increased secretion of IFNγ, TNF-α, and IL-10. Preliminary data showed that T cells primed by Mo-DCs activated with p14ARF+IFNβ-transduced SK-MEL-147 cells were able to induce the loss of viability of fresh non-transduced SK-MEL-147 cells, suggesting the induction of a specific cytotoxic population that recognized and killed SK-MEL-147 cells. Collectively, our results indicate that p14ARF and IFNβ delivered by our adenoviral system induced oncolysis in human melanoma cells accompanied by adaptive immune response activation and regulation.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-γ and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.